Stem cells of the islets of langerhans and their use in treating diabetes mellitus

ABSTRACT

Methods and compositions are described for the treatment of type I insulin-dependent diabetes mellitus and other conditions using newly identified stem cells that are capable of differentiation into a variety of pancreatic islet cells, including insulin-producing beta cells, as well as hepatocytes. Nestin and ABCG2 have been identified as molecular markers for pancreatic stem cells, while cytokeratin-19 serves as a marker for a distinct class of islet ductal cells. Methods are described whereby nestin and/or ABCG2-positive stem cells can be isolated from pancreatic islets and cultured to obtain further stem cells or pseudo-islet like structures. Methods for ex vivo differentiation of the pancreatic stem cells are disclosed. Methods are described whereby pancreatic stem cells can be isolated, expanded, and transplanted into a patient in need thereof, either allogeneically, isogeneically or xenogenically, to provide replacement for lost or damaged insulin-secreting cells or other cells.

PRIORITY

[0001] The present application claims priority to U.S. application Ser.No. 09/731,261, filed Dec. 6, 2000 and U.S. application Ser. No.09/963,875, filed Sep. 26, 2001.

TECHNICAL FIELD OF THE INVENTION

[0002] The invention is related to the field of stem cells and theirdifferentiation. In particular, it is related to the field of beta cellsof the islets of Langerhans in the pancreas and nestin positive liverstem cells and their differentiation from stem cells or progenitorcells, and the use of pancreatic stem cells, progenitor cells, anddifferentiated beta cells or nestin positive liver stem cells orprogenitor cells in transplantation.

BACKGROUND OF THE INVENTION

[0003] The invention was made at least in part using U.S. governmentfunds, grants DK30457, DK30834, DK55365, DK60125, awarded by theNational Institutes of Health, and therefore the U.S. government mayretain certain rights in the invention.

[0004] The origin of pancreatic islet cells, both during embryonicdevelopment and in a mature mammal, has remained uncertain despiteintensive study. Certain ductal epithelial cells are capable of eitherdifferentiation or transdifferentiation to form beta cells and othercell types found in mature islets (Bouwens, 1998). Ductal cells fromisolated islets can proliferate in culture and, if transplanted into ananimal, can differentiate into functional beta cells (Cornelius et al.,1997).

[0005] It has been demonstrated that exendin-4, a long acting GLP-1agonist, stimulates both the differentiation of β-cells from ductalprogenitor cells (neogenesis) and proliferation of β-cells whenadministered to rats. In a partial pancreatectomy rat model of type 2diabetes, the daily administration of exendin-4 for 10 days postpancreatectomy attenuated the development of diabetes. It has also beendemonstrated that exendin-4 stimulates the regeneration of the pancreasand expansion of β-cell mass by neogenesis and proliferation of β-cells(Xu et al., 1999, Diabetes, 48:2270-2276).

[0006] Ramiya et al. have demonstrated that islets generated in vitrofrom pluripotent stem cells isolated from the pancreatic ducts of adultprediabetic non-obese diabetic (NOD) mice differentiate to form glucoseresponsive islets that can reverse insulin-dependent diabetes afterbeing implanted, with or without encapsulation, into diabetic NOD mice(Ramiya et al., 2000, Nature Med., 6:278-282).

[0007] The insulinotropic hormone glucagon-like peptide (GLP)-1 which isproduced by the intestine, enhances the pancreatic expression of thehomeodomain transcription factor IDX-1 that is critical for pancreasdevelopment and the transcriptional regulation of the insulin gene.Concomitantly, GLP-1 administered to diabetic mice stimulates insulinsecretion and effectively lowers their blood sugar levels. GLP-1 alsoenhances β-cell neogenesis and islet size (Stoffers et al., 2000,Diabetes, 49:741-748).

[0008] Ferber et al. have demonstrated that adenovirus-mediated in vivotransfer of the PDX-1 (also known as IDX-1) transgene to mouse liverresults in the transconversion of a hepatocyte subpopulation towards aβ-cell phenotype. It has been demonstrated that after intravenousinfusion of mice with the PDX-1 adenoviral vector, up to 60% ofhepatocytes synthesized PDX-1. Although 60% of liver cells becameinfected with the adenovirus and expressed PDX-1, only a subset of 5-8%of these cells turned into insulin expressing beta cells. Theconcentration of immunoreactive insulin was increased in the liver andserum of treated mice. Mice treated with PDX-1 survivestreptozotocin-induced diabetes, and can even normalize glycemia (Ferberet al., 2000, Nature Med., 6:568-572).

[0009] While ductal cell cultures obtained from isolated isletsapparently contain cells that can give rise to insulin-secreting cells,it has remained unclear whether those cells represent true stem cells ormerely ductal epithelial cells undergoing transdifferentiation. Even ifsuch preparations contain genuine stem cells, it is unknown whatfraction represent stem cells and what contaminating cell types may bepresent. There is a need in the art for the isolation of specific celltypes from pancreatic tissue, the cell types being characterized as stemcells using molecular markers and demonstrated to be pluripotent and toproliferate long-term.

[0010] Pluripotent stem cells that are capable of differentiating intoneuronal and glial tissues have been identified in brain. Neural stemcells specifically express nestin, an intermediate filament protein(Lendahl et al., 1990; Dahlstrand et al., 1992). Nestin is expressed inthe neural tube of the developing rat embryo at day E11, reaches maximumlevels of expression in the cerebral cortex at day E16, and decreases inthe adult cortex, becoming restricted to a population of ependymal cells(Lendahl et al., 1990). Developing neural and pancreatic islet cellsexhibit phenotypic similarities characterized by common cellularmarkers.

[0011] The invention relates to a population of pancreatic isletstem/progenitor cells (IPCs) that are similar to neural and hepatic stemcells and differentiate into islet α-cells (glucagon) and β-cells(insulin). The invention also relates to nestin-positive liver cells.IPCs according to the invention are immunologicallysilent/immunoprivileged and are recognized by a transplant recipient asself. The IPCs according to the invention can be used for engraftmentacross allogeneic and xenogeneic barriers.

[0012] There is a need in the art for a method of engrafting stem cellsacross allogeneic and xenogeneic barriers.

[0013] There is also a need in the art for a method of treating type Idiabetes mellitus wherein islets, nestin-positive pancreatic stem cellsor nestin-positive liver stem cells are transferred into a recipientacross allogeneic or xenogeneic barriers and graft rejection does notoccur.

[0014] There is also a need in the art for a method of transplantationinto a mammal wherein islets, nestin-positive pancreatic stem cells ornestin-positive liver stem cells are transferred into a recipient acrossallogeneic or xenogeneic barriers and graft rejection does not occur.

SUMMARY OF THE INVENTION

[0015] It is an object of the invention to provide mammalian pancreaticor liver stem cells for use in treating diabetes mellitus and otherdisorders. It is also an object of the invention to provide methods foridentifying, localizing, and isolating pancreatic stem cells. It is afurther object of the invention to provide methods for differentiatingpancreatic stem cells to obtain cells that produce insulin and otherhormones. It is also an object of the invention to provide methods fortransplantation into a mammal that utilize mammalian pancreatic or liverstem cells. These and other objects of the invention are provided by oneor more of the embodiments described below.

[0016] One embodiment of the invention provides a method of treating apatient with diabetes mellitus. A nestin-positive pancreatic stem cellis isolated from a pancreatic islet of a donor. In another embodiment, apancreatic stem cell that is positive for at least one of ABCG2, Oct3/4,GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunitsα6 and β1, C-kit, MDR-1, SUR-1, Kir 6.2 and/or does not express one orall of CD34, CD45, CD133, MHC class I and MHC class II is isolated froma pancreatic islet of a donor. In another embodiment, a pancreatic stemcell that is positive for SST-R2, SST-R3 and/or SST-R4 is isolated froma pancreatic islet of a donor. The stem cell is transferred into thepatient, where it differentiates into an insulin-producing cell.

[0017] Another embodiment provides another method of treating a patientwith diabetes. A nestin-positive pancreatic stem cell is isolated from apancreatic islet of a donor and expanded ex vivo to produce a progenitorcell. In another embodiment, a pancreatic stem cell that is positive forat least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2,SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one or all ofCD34, CD45, CD133, MHC class I and MHC class II is isolated from apancreatic islet of a donor and expanded ex vivo to produce a progenitorcell. The progenitor cell is transferred into the patient, where itdifferentiates into an insulin-producing beta cell. Another embodimentprovides still another method of treating a diabetes patient. Anestin-positive pancreatic stem cell is isolated from a pancreatic isletof a donor and expanded to produce a progenitor cell. In anotherembodiment, a pancreatic stem cell that is positive for at least one ofABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin,Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4,SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45, CD133,MHC class I and MHC class II is isolated from a pancreatic islet of adonor and expanded to produce a progenitor cell. The progenitor cell isdifferentiated in culture to form pseudo-islet like aggregates that aretransferred into the patient.

[0018] Another embodiment provides another method of treating a patientwith diabetes mellitus. A nestin-positive pancreatic stem cell isisolated from a pancreatic islet of a donor and cultured ex vivo toproduce a progenitor cells. In another embodiment, a pancreatic stemcell that is positive for at least one of ABCG2, Oct3/4, GLP-1 receptor,latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit,MDR-1, SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not expressone or all of CD34, CD45, CD133, MHC class I and MHC class II isisolated from a pancreatic islet of a donor and cultured ex vivo toproduce a progenitor cell. The progenitor cell is transferred into thepatient, where it differentiates into an insulin-producing beta cell.

[0019] In these embodiments, the patient can also serve as the donor ofthe pancreatic islet tissue, providing an isograft of cells ordifferentiated tissue.

[0020] In another preferred embodiment, prior to the step oftransferring, the stem cell is treated ex vivo with an agent selectedfrom the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF,GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1,betacellulin, activin A, TGF-β, and combinations thereof.

[0021] In another preferred embodiment, the step of transferring isperformed via endoscopic retrograde injection or injection into thepancreatic artery.

[0022] In another preferred embodiment, the method of treating a patientwith diabetes mellitus additionally comprises the step of treating thepatient with an immunosuppressive agent.

[0023] In another preferred embodiment, the immunosuppressive agent isselected from the group consisting of FK-506, cyclosporin, and GAD65antibodies.

[0024] Another embodiment provides a method of isolating a stem cellfrom a pancreatic islet of Langerhans. A pancreatic islet is removedfrom a donor, and cells are cultured from it. A nestin-positive stemcell clone is selected from the culture. In another embodiment, a cloneof pancreatic stem cells that is positive for at least one of ABCG2,Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrinsubunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2and/or do not express one or all of CD34, CD45, CD133, MHC class I andMHC class II is selected from the culture. Optionally, the islet isfirst purged of non-islet cells by culturing in a vessel coated withconcanavalin A, which binds the non-islet cells.

[0025] In a preferred embodiment, the method of isolating a stem cellfurther comprises the additional step of expanding the clone bytreatment with an agent selected from the group consisting of EGF,bFGF-2, high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleicacid molecule encoding IDX-1, betacellulin, activin A, TGF-β, andcombinations thereof.

[0026] A further embodiment provides a method of inducing thedifferentiation of a nestin-positive pancreatic stem cell into apancreatic progenitor cell. Another embodiment provides a method ofinducing the differentiation of a pancreatic stem cell that is positivefor at least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2,SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one or all ofCD34, CD45, CD133, MHC class I and MHC class II . As used herein,“differentiation” refers to the process by which a cell undergoes achange to a particular cell type, e.g. to a specialized cell type. Thestem cell is treated with an agent selected from the group consisting ofEGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleic acid moleculeencoding IDX-1, GLP-1, exendin-4, betacellulin, activin A, TGF-β, andcombinations thereof. The stem cell subsequently differentiates into apancreatic progenitor cell.

[0027] In a preferred embodiment, the pancreatic progenitor subsequentlyforms pseudo-islet like aggregates.

[0028] Yet another embodiment provides an isolated, nestin-positivehuman pancreatic or liver stem cell. Yet another embodiment provides anisolated pancreatic stem cell that is positive for at least one ofABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin,Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4,SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45, CD133,MHC class I and MHC class II. In versions of this embodiment, the stemcell differentiates into either a beta cell, an alpha cell, apseudo-islet like aggregate, or a hepatocyte. In versions of thisembodiment, the stem cell is immunoprivileged. In versions of thisembodiment, the stem cell does not express class I MHC antigens. Inversions of this embodiment, the stem cell does not express class II MHCantigens. In versions of this embodiment, the stem cell does not expressclass I or class II MHC antigens.

[0029] Still another embodiment provides a method of identifying apancreatic cell as a stem cell. A cell is contacted with a labelednestin-specific antibody. If the cell becomes labeled with the antibody,then the cell is identified as a stem cell. In another embodiment, apancreatic stem cell is identified as a stem cell by contacting the cellwith an antibody specific for any of ABCG2, Oct3/4, GLP-1 receptor,latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit,MDR-1, SST-R2, SST-R3, SST-R4, SUR-1 or Kir 6.2 wherein a cell thatbecomes labeled with an antibody is identified as a stem cell. Optionaladditional steps include contacting the cell with an antibody tocytokeratin 19 and an antibody to collagen IV; the cell is identified asa stem cell if it does not become labeled with either the cytokeratin 19or the collagen IV antibody.

[0030] In another embodiment, a pancreatic stem cell is identified as astem cell by contacting the cell with an antibody specific for any ofCD34, CD35, CD133, MHC Class I and MHC Class II, wherein the cell isidentified as a stem cell if it does not become labeled with any of theantibodies.

[0031] Another embodiment provides a method of inducing anestin-positive pancreatic stem cell to differentiate into hepatocytes.The nestin-positive pancreatic stem cell is treated with an effectiveamount of an agent that induces the stem cell to differentiate intohepatocytes or into progenitor cells that differentiate intohepatocytes. In a preferred embodiment, the agent is cyclopamine.Another embodiment provides a method of inducing a pancreatic stem cellthat is positive for at least one of ABCG2, Oct3/4, GLP-1 receptor,latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit,MDR-1, SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not expressone or all of CD34, CD45, CD133, MHC class I and MHC class II todifferentiate into hepatocytes by treatment with an effective amount ofan agent that induces the stem cell to differentiate into hepatocytes orinto progenitor cells that differentiate into hepatocytes.

[0032] Yet another embodiment provides a method of treating a patientwith liver disease. A nestin-positive pancreatic stem cell is isolatedfrom a pancreatic islet of a donor and transferred into the patient,where the stem cell differentiates into a hepatocyte. In anotherembodiment, a pancreatic stem cell that is positive for at least one ofABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin,Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3, SST-R4,SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45, CD133,MHC class I and MHC class II is isolated from a pancreatic islet of adonor and transferred into the patient where the stem celldifferentiates into a hepatocyte.

[0033] In a related embodiment, the stem cell is expanded ex vivo to aprogenitor cell, which is transferred into the patient and furtherdifferentiates into a hepatocyte.

[0034] In another related embodiment, the stem cell is differentiated exvivo to a progenitor cell, which is transferred into the patient andfurther differentiates into a hepatocyte. In another related embodiment,the stem cell is differentiated ex vivo into hepatocytes, which aretransplanted into the patient.

[0035] In these embodiments, the patient can also serve as the donor ofthe pancreatic islet tissue, providing an isograft of cells ordifferentiated tissue.

[0036] Yet another embodiment provides an isolated, nestin-positivehuman liver stem cell. In versions of this embodiment, the stem cell isimmunoprivileged. In versions of this embodiment the stem cell does notexpress class I MHC antigens. In versions of this embodiment, the stemcell does not express class II MHC antigens. In versions of thisembodiment, the stem cell does not express class I or class II MHCantigens.

[0037] Yet another embodiment provides an isolated, nestin-positivehuman stem cell that is not a neural stem cell, that is capable oftransplant into an animal without causing graft versus host rejection.In versions of this embodiment, the stem cell is not majorhistocompatibility complex class I or class I restricted. Still anotherembodiment provides an isolated human stem cell that is positive for atleast one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1,Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3,SST-R4, SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45,CD133, MHC class I and MHC class II, that is not a neural stem cell,that is capable of transplant into an animal without causing graftversus host rejection.

[0038] A “stem cell” as used herein is a undifferentiated cell which iscapable of essentially unlimited propagation either in vivo or ex vivoand capable of differentiation to other cell types. This can be tocertain differentiated, committed, immature, progenitor, or mature celltypes present in the tissue from which it was isolated, or dramaticallydifferentiated cell types, such as for example the erythrocytes andlymphocytes that derive from a common precursor cell, or even to celltypes at any stage in a tissue completely different from the tissue fromwhich the stem cell is obtained. For example, blood stem cells maybecome brain cells or liver cells, neural stem cells can become bloodcells, such that stem cells are pluripotential, and given theappropriate signals from their environment, they can differentiate intoany tissue in the body.

[0039] In one embodiment, a “stem cell” according to the invention isimmunologically blinded or immunoprivileged. As used herein,“immunologically blinded” or “immunoprivileged” refers to a cell thatdoes not elicit an immune response. As used herein, an “immune response”refers to a response made by the immune system to a foreign substance.An immune response, as used herein, includes but is not limited totransplant or graft rejection, antibody production, inflammation, andthe response of antigen specific lymphocytes to antigen. An immuneresponse is detected, for example, by determining if transplantedmaterial has been successfully engrafted or rejected, according tomethods well-known in the art, and as defined herein in the sectionentitled, “Analysis of Graft Rejection”. In one embodiment, an“immunogically blinded stem cell” or an “immunoprivileged stem cell”according to the invention can be allografted or xenografted withouttransplant rejection, and is recognized as self in the transplantrecipient or host.

[0040] Transplanted or grafted material can be rejected by the immunesystem of the transplant recipient or host unless the host isimmunotolerant to the transplanted material or unless immunosupressivedrugs are used to prevent rejection.

[0041] As used herein, a host that is “immunotolerant”, according to theinvention, fails to mount an immune response, as defined herein. In oneembodiment, a host that is “immunotolerant” does not reject or destroytransplanted material. In one embodiment, a host that is“immunotolerant” does not respond to an antigen by producing antibodiescapable of binding to the antigen.

[0042] As used herein, “rejection” refers to rejection of transplantedmaterial by the immune system of the host. In one embodiment, “rejectionmeans an occurrence of more than 90% cell or tissue necrosis of thetransplanted material in response to the immune response of the host. Inanother embodiment, “rejection” means a decrease in the viability suchthat the viability of the transplanted material is decreased by 90% ormore as compared to the viability of the transplanted material prior totransplantation, in response to the immune response of the host. Adecrease in viability can be determined by methods well known in theart, including but not limited to trypan blue exclusion staining. Inanother embodiment, “rejection” means failure of the transplantedmaterial to proliferate. Proliferation can be measured by methods knownin the art including but not limited to hematoxylin/eosin staining. Theoccurrence of transplant rejection and/or the speed at which rejectionoccurs following transplantation will vary depending on factors,including but not limited to the transplanted material (i.e., the celltype, or the cell number) or the host (i.e., whether or not the host isimmunotolerant and/or has been treated with an immunosuppressive agent.As used herein, “graft versus host rejection” or “graft versus hostresponse” refers to a cell-mediated reaction in which T-cells of thetransplanted material react with antigens of the host.

[0043] As used herein, “host versus graft rejection” or “host versusgraft response” refers to a cell-mediated reaction in which cells of thehost's immune system attack the foreign grafted or transplantedmaterial.

[0044] In another embodiment of the invention, an immune response hasoccurred if production of a specific antibody (for example an antibodythat binds specifically to an antigen on the transplanted material, oran antibody that binds specifically to the foreign substance or aproduct of the foreign substance) is detected by immunological methodswell-known in the art, including but not limited to ELISA,immunostaining, immunoprecipitation and Western Blot analysis.

[0045] Stem cells express morphogenic or growth hormone receptors on thecell surface, and can sense, for example, injury-related factors thenlocalize to and take residence at sites of tissue injury, or sense theirlocal microenvironment and differentiate into the appropriate cell type.

[0046] “Essentially unlimited propagation” can be determined, forexample, by the ability of an isolated stem cell to be propagatedthrough at least 50, preferably 100, and even up to 200 or more celldivisions in a cell culture system. Stem cells can be “totipotent,”meaning that they can give rise to all the cells of an organism as forgerm cells. “Totipotent” also means the fertilized egg that can giverise to both embryo and trophoblast. Stem cells can also be“pluripotent,” meaning that they can give rise to many different celltypes, but not all the cells of an organism. “Pluripotent” also meansgives rise to embryo only and not to trophoblast. When a stem celldifferentiates it generally gives rise to a more adult cell type, whichmay be a partially differentiated cell such as a progenitor cell, adifferentiated cell, or a terminally differentiated cell. Stem cells canbe highly motile.

[0047] “Nestin” refers to an intermediate filament protein having asequence disclosed in Genbank Access No. X65964 (FIG. 7).

[0048] “ABCG2” refers to ATP-binding cassette multidrug resistancetransporter G2 having a sequence disclosed in Genbank Access No.XM_(—)032424 (FIG. 18). One of skill in the art will recognize thatequivalents exist wherein the DNA sequence encoding ABCG2 may vary, butwherein the encoded amino acid sequence remains the same.

[0049] “Oct3/4” refers to a POU/Homeodomain transcription factor havinga sequence disclosed in Genbank Access No. NM 013633.

[0050] “GLP-1R” refers to the glucagon-like peptide-1-receptor encodedby the nucleic acid sequence shown in FIG. 21 (GenBank Accession No.U01156), and having the amino acid sequence also shown in FIG. 17(GenBank Accession No. U01156). One of skill in the art will recognizethat equivalents exist wherein the DNA sequence encoding GLP-1R mayvary, but wherein the encoded amino acid sequence remains the same.

[0051] “Latrophilin (type 2)” refers to a G protein-coupled receptorhaving a sequence disclosed in Genbank Access No. AJ131581.

[0052] “Hes-1” refers to a bHLH transcription factor having a sequencedisclosed in Genbank Access No. NM_(—)005524.

[0053] Integrins are integral cell-surface proteins composed of an alphachain and a beta chain. A given chain may combine with multiple partnersresulting in different integrins. For example, alpha 6 may combine withbeta 4 in the integrin referred to as TSP180, or with beta 1 in theintegrin VLA-6.

[0054] The integrin subunit “α6” refers to the human integrin α6 cDNAhaving a sequence disclosed in Genbank Access No. NM_(—)000210.

[0055] The integrin subunit “β1” refers to the human β1 cDNA having asequence disclosed in Genbank Access No.: X07979.

[0056] “c-Kit” refers to a cell surface receptor tyrosine kinase havinga sequence disclosed in Genbank Access No. X06182.

[0057] “MDR-1” refers to the multidrug resistance transporter 1 having asequence disclosed in Genbank Access No. NM_(—)000927.

[0058] “SST-R2” SST-R3” and “SST-R4” refer to somatostatin receptorshaving a sequence disclosed in Genbank Access Nos. XM_(—)085745(SSTR-2); NM_(—)001051 (SSTR-3); XM_(—)009594 (SSTR-4).

[0059] “SUR-1” refers to a human sulfonylurea receptor having a sequencedisclosed in Genbank Access No. XM_(—)042740.

[0060] “Kir 6.2” refers to a human inward rectifier potassium channelhaving a sequence disclosed in Genbank Access No. AF021139.

[0061] “CD34” refers to a transmembrane glycoprotein having a sequencedisclosed in Genbank Access No. NM_(—)001773.

[0062] “CD45” refers to the leukocyte common antigen having a sequencedisclosed in Genbank Access No. Y00638.

[0063] “CD133” refers to a five-transmembrane hematopoietic stem cellantigen having a sequence disclosed in Genbank Access No. NM_(—)006017.

[0064] A “pancreatic” stem cell means a stem cell that has been isolatedfrom pancreatic tissue and/or a cell that has all of the characteristicsof: nestin-positive staining, nestin gene expression, cytokeratin-19negative staining, long-term proliferation in culture, and the abilityto differentiate into pseudo-islets in culture.

[0065] A “pancreatic” stem cell also refers to a stem cell that ispositive for at least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin(type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1,SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45, CD133,MHC class I and MHC class II.

[0066] In one embodiment, a “pancreatic” stem cell also refers to a stemcell that is positive for SST-R2, SST-R3 and/or SST-R4.

[0067] A “liver” stem cell means a stem cell that has been isolated fromliver tissue and/or a cell that has all of the characteristics of:nestin -positive staining, nestin gene expression, and long-termproliferation in culture.

[0068] A “progenitor cell” is a cell that is derived from a stem cell bydifferentiation and is capable of further differentiation to more maturecell types.

[0069] As used herein, the term “insulin-producing beta cell” refers toany cell which can produce and secrete insulin in a similar amount tothat produced and secreted by a beta cell of the islets of Langerhans inthe human pancreas. Preferably, the secretion of insulin by aninsulin-producing beta cell is also regulated in a similar fashion tothe regulation of insulin secretion by a human beta cell in situ; forexample, insulin secretion should be stimulated by an increase in theglucose concentration in the solution surrounding the insulin-producingbeta cell.

[0070] “Pseudo-islet like” aggregates are artificial aggregates ofinsulin-secreting cells which resemble in form and function the isletsof Langerhans of the pancreas. Pseudo-islet like aggregates are createdex vivo under cell culture conditions. They are approximately 50-150 μmin diameter (compared to an average diameter of 100 μm for in situislets) and spheroid in form.

[0071] “Isolating” a stem cell refers to the process of removing a stemcell from a tissue sample and separating away other cells which are notstem cells of the tissue. An isolated stem cell will be generally freefrom contamination by other cell types and will generally have thecapability of propagation and differentiation to produce mature cells ofthe tissue from which it was isolated. However, when dealing with acollection of stem cells, e.g., a culture of stem cells, it isunderstood that it is practically impossible to obtain a collection ofstem cells which is 100% pure. Therefore, an isolated stem cell canexist in the presence of a small fraction of other cell types which donot interfere with the utilization of the stem cell for analysis orproduction of other, differentiated cell types. Isolated stem cells willgenerally be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%,or 99% pure. Preferably, isolated stem cells according to the inventionwill be at least 98% or at least 99% pure.

[0072] A stem cell is “expanded” when it is propagated in culture andgives rise by cell division to other stem cells and/or progenitor cells.Expansion of stem cells may occur spontaneously as stem cellsproliferate in a culture or it may require certain growth conditions,such as a minimum cell density, cell confluence on the culture vesselsurface, or the addition of chemical factors such as growth factors,differentiation factors, or signaling factors.

[0073] A stem cell, progenitor cell, or differentiated cell is.“transplanted” or “introduced” into a mammal when it is transferred froma culture vessel into a patient. Transplantation, as used herein, caninclude the steps of isolating a stem cell according to the inventionand transferring the stem cell into a mammal or a patient.Transplantation can involve transferring a stem cell into a mammal or apatient by injection of a cell suspension into the mammal or patient,surgical implantation of a cell mass into a tissue or organ of themammal or patient, or perfusion of a tissue or organ with a cellsuspension. The route of transferring the stem cell or transplantation,will be determined by the need for the cell to reside in a particulartissue or organ and by the ability of the cell to find and be retainedby the desired target tissue or organ. In the case where a transplantedcell is to reside in a particular location, it can be surgically placedinto a tissue or organ or simply injected into the bloodstream if thecell has the capability to migrate to the desired target organ.

[0074] Transplantation, as used herein, can include the steps ofisolating a stem cell according to the invention, and culturing andtransferring the stem cell into a mammal or a patient. Transplantation,as used herein, can include the steps of isolating a stem cell accordingto the invention, differentiating the stem cell, and transferring thestem cell into a mammal or a patient. Transplantation, as used herein,can include the steps of isolating a stem cell according to theinvention, differentiating and expanding the stem cell and transferringthe stem cell into a mammal or a patient.

[0075] Treatment with an immunosuppressive agent can be accomplished byadministering to a patient in need thereof any agent which prevents,delays the occurrence of or decreases the intensity of the desiredimmune response, e.g., rejection of a transplanted cell, tissue, ororgan.

[0076] As used herein, “immunosuppression” refers to prevention of theimmune response (for example by the administration of an“immunosuppresive agent”, as defined herein) such that an “immuneresponse”, as defined herein, is not detectable. As used herein,“prevention” of an immune response means an immune response is notdetectable. An immune response (for example, transplant rejection orantibody production) is detected according to methods well-known in theart and defined herein.

[0077] “Immunosuppression” according to the invention also means a delayin the occurrence of the immune response as compared to any one of atransplant recipient that has not received an immunosuppresive agent, ora transplant recipient that has been transplanted with material that isnot “immunologically blinded” or “immunoprivileged”, as defined herein.A delay in the occurrence of an immune response can be a short delay,for example 1 hr-10 days, i.e., 1 hr, 2, 5 or 10 days. A delay in theoccurrence of an immune response can also be a long delay, for example,10 days-10 years (i.e., 30 days, 60 days, 90 days, 180 days, 1, 2, 5 or10 years).

[0078] “Immunosuppression” according to the invention also means adecrease in the intensity of an immune response. According to theinvention, the intensity of an immune response can be decreased suchthat it is 5-100%, preferably, 25-100% and most preferably 75 -100% lessthan the intensity of the immune response of any one of a transplantrecipient that has not received an immunosuppresive agent, or atransplant recipient that has been transplanted with material that isnot “immunologically blinded” or “immunoprivileged”, as defined herein.The intensity of an immune response can be measured by determining thetime point at which transplanted material is rejected. For example, animmune response comprising rejection of transplanted material at day 1,post-transplantation, is of a greater intensity than an immune responsecomprising the rejection of transplanted material at day 30,post-transplantation. The intensity of an immune response can also bemeasured by quantitating the amount of a particular antibody capable ofbinding to the transplanted material, wherein the level of antibodyproduction correlates directly with the intensity of the immuneresponse. Alternatively, the intensity of an immune response can bemeasured by determining the time point at which a particular antibodycapable of binding to the transplanted material is detected.

[0079] Various strategies and agents can be utilized forimmunosuppression. For example, the proliferation and activity oflymphocytes can be inhibited generally with agents such as, for example,FK-506, or cyclosporin or other immunosuppressive agents. Anotherpossible strategy is to administer an antibody, such as an anti-GAD65monoclonal antibody, or another compound which masks a surface antigenon a transplanted cell and therefore renders the cell practicallyinvisible to the immune system of the host.

[0080] An “immunosuppressive agent” is any agent that prevents, delaysthe occurrence of or reduces the intensity of an immune reaction againsta foreign cell in a host, particularly a transplanted cell. Preferredare immunosuppressive agents which suppress cell-mediated immuneresponses against cells identified by the immune system as non-self.Examples of immunosuppressive agents include but are not limited tocyclosporin, cyclophosphamide, prednisone, dexamethasone, methotrexate,azathioprine, mycophenolate, thalidomide, FK-506, systemic steroids, aswell as a broad range of antibodies, receptor agonists, receptorantagonists, and other such agents as known to one skilled in the art.

[0081] A “mitogen” is any agent that stimulates mitosis and cellproliferation of a cell to which the agent is applied.

[0082] A “differentiation factor” is any agent that causes a stem cellor progenitor cell to differentiate into another cell type.Differentiation is usually accomplished by altering the expression ofone or more genes of the stem cell or progenitor cell and results in thecell altering its structure and function.

[0083] A “signaling factor” as used herein is an agent secreted by acell which has an effect on the same or a different cells. For example,a signaling factor can inhibit or induce the growth, proliferation, ordifferentiation of itself, neighboring cells, or cells at distantlocations in the organism. Signaling factors can, for example, transmitpositional information in a tissue, mediate pattern formation, or affectthe size, shape and function of various anatomical structures.

[0084] As used herein, a mammal refers to any mammal including but notlimited to human, mouse, rat, sheep, monkey, goat, rabbit, hamster,horse, cow or pig.

[0085] A “non-human mammal”, as used herein, refers to any mammal thatis not a human.

[0086] As used herein, “allogeneic” refers to genetically differentmembers of the same species.

[0087] As used herein, “isogeneic” refers to of an identical geneticconstitution.

[0088] As used herein, “xenogeneic” refers to members of a differentspecies.

[0089] As used herein, “culturing” refers to propagating or nurturing acell, collection of cells, tissue, or organ, by incubating for a periodof time in an environment and under conditions which support cellviability or propagation. Culturing can include one or more of the stepsof expanding and proliferating a cell, collection of cells, tissue, ororgan according to the invention.

[0090] The invention also provides for a pharmaceutical compositioncomprising the isolated stem cells of the invention admixed with aphysiologically compatible carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0091]FIGS. 1A and 1B show dual fluorescence immunocytochemical stainingof rat pancreatic islets at embryonic day 16 (FIG. 1A) and at day 60after birth (FIG. 1B). Immunostaining with an antibody for nestin isshown in white (red in the original, with Cy3 as fluorophore) and withan antibody for insulin is shown in grey (green in the original, withCy2 as fluorophore).

[0092]FIG. 2 shows the result of RT-PCR performed using mRNA obtainedfrom 50 rat islets. Forward and reverse primers are indicated. Thesingle band of 834 bp was sequenced and identified substantially as thesequence for nestin.

[0093]FIG. 3 shows nestin-positive cells that have proliferated out froma cultured rat islet.

[0094]FIG. 4 shows the development of islet like structures in culture.

[0095]FIG. 5 shows the results of RT-PCR analysis of islet-likestructures generated in culture. Expression of NCAM and cytokeratin-19(CK19) was detected.

[0096]FIG. 6 shows the stimulation of nestin mRNA expression by highglucose. APRT was examined as a control.

[0097]FIG. 7 is the amino acid and nucleotide sequences of nestin.

[0098]FIG. 8 depicts expression of the neural stem cell-specific markernestin in a distinct cell population within pancreatic islets asdetermined by immunocytochemistry or RT-PCR.

[0099]FIG. 9 depicts characterization of nestin in stem cells isolatedfrom the pancreas by immunocytochemistry and RT-PCR.

[0100]FIG. 10 depicts expression of homeodomain protein IDX-1 andproglucagon in human islet-like clusters derived from nestin-positiveislet progenitor cells (NIPs).

[0101]FIG. 11 demonstrates localization of nestin-positive cells tolocalized regions of the ducts of the rat pancreas.

[0102]FIG. 12 depicts alternative models for the origin of pancreaticduct cells that are progenitors of islet endocrine cells.

[0103]FIG. 13A and B depicts immunofluorescent staining of nestinpositive liver stem cells.

[0104]FIG. 14 depicts the sequential appearance of transcription factorsduring development of the murine endocrine pancreas.

[0105]FIG. 15 depicts expression of neuroendocrine, exocrine pancreaticand hepatic markers in human NIP cultures containing stem cells.

[0106]FIG. 16 depicts expression of proglucagon and insulin mRNA asdetermined by RT-PCR and insulin secretion.

[0107]FIG. 17 depicts NIP markers.

[0108]FIG. 18 depicts the nucleic acid(a) and amino acid(b) sequence ofhuman ABCG2.

[0109]FIG. 19a depicts expression of the ATP-binding cassettetransporter ABCG2 by RT-PCR and Southern blot hybridization.

[0110]FIG. 19b demonstrate that nestin-positive islet derived progenitorcells (NIPs). include a significant number of SP cells as demonstratedby Hoechst 33342 staining.

[0111]FIG. 19c demonstrates that dye efflux from SP cells is inhibitedin the presence of verapamil. SP-gated cells are 2.1% of the totalnumber of analyzed cells in B versus 0.1% in C.

[0112]FIG. 20 demonstrates that SP cells isolated by FACS co-expresshigh levels of ABCG2, MDR1 and nestin. FIG. 20A depicts that SP cellsand non-SP control cells were isolated by FACS after Hoechst 33342staining (R1 and R2, respectively). FIG. 20B demonstrates that the cellswere analyzed for the expression of ABCG2, MDR1, nestin, and GAPDH RNAby RT-PCR. The identity of the PCR products for ABCG2 and MDR1 wasconfirmed by Southern blot hybridization.

[0113]FIG. 21 shows the nucleic acid (21A) and amino acid (21B) sequenceof the human GLP-1R.

[0114]FIG. 22 shows the expression of GLP-1R on pancreatic islet-derivedStem/progenitor cells. Panel A shows the immunocytochemical detection ofGLP-1R (Cy-3) and nuclei stained with DAPI. Panel B shows RT-PCR of RNAprepared from NIPS with primers specific for GLP-1R.

[0115]FIG. 23 shows GLP-1 (7-36) amide and tolbutamide stimulation ofCa²⁺ influx in nestin-positive NIPs.

[0116]FIG. 24 shows immunohistochemical staining of NIPs demonstratingGLP-1 induced differentiation. Panel A shows immunohistochemicalidentification of nestin and insulin. Panel B shows nestin and insulinimmunohistochemical staining after cells were challenged with GLP-1.

[0117]FIG. 25 shows the levels of insulin secretion from NIP cultureschallenged with GLP-1.

[0118]FIG. 26 shows the immunohistochemical analysis of insulinexpression in NIP cultures transfected with human Idx-1.

DETAILED DESCRIPTION OF THE INVENTION

[0119] The present inventors have identified and isolated a specialsubclass of ductal cells from the islets of Langerhans of mammalianpancreas that have the functional and molecular characteristics of stemcells. In particular, these newly discovered pancreatic stem cells arecharacterized inter alia by one or more (and preferably all of):nestin-positive staining, nestin gene expression, GLP-1R-positivestaining, GLP-1R gene expression, ABCG2 positive staining, ABCG2 geneexpression,Oct3/4 positive staining, Oct3/4 gene expression, latrophilin(type 2) positive staining, latrophilin (type 2) gene expression, Hes-1positive staining, Hes-1 gene expression, Integrin subunits α6 and β1positive staining, Integrin subunits α6 and β1 gene expression, C-kitpositive staining, C-kit gene expression, MDR-1 positive staining, MDR-1gene expression, SST-R, 2, 3, 4 positive staining, SST-R, 2, 3, 4 geneexpression, SUR-1 positive staining, SUR-1 gene expression, Kir 6.2positive staining, Kir 6.2 gene expression CD34 negative staining, CD45negative staining, CD133 negative staining, MHC class I negativestaining, MHC class II negative staining,cytokeratin-19 negativestaining, long-term proliferation in culture, and the ability todifferentiate into pseudo-islets in culture. The present inventors havealso identified liver cells that exhibit nestin-positive staining.

[0120] In one embodiment, the invention provides stem cells for avariety of applications, including but not limited to cellularreplacement therapy for type I insulin-dependent diabetes and otherforms of diabetes as well as the development of research tools to studythe onset and progression of various diabetic conditions, hormonalabnormalities, and genetic diseases or conditions, such as theassociation of polymorphisms with particular physiologic or pathologicstates. The stem cells of the invention can also be used to carry outgene therapy of endocrine pancreatic or other tissues in isograft,allograft or xenograft transplantations. Further, the stem cellsdescribed herein can be used to produce recombinant cells, artificialtissues, and replacement organs in culture. They can also be used forthe ex vivo production of insulin and other hormones. Molecularcharacteristics of pancreatic stem cells discovered by the inventors,such as nestin-positive, GLP-1R-positive, ABCG2 positive staining,Oct3/4 positive, latrophilin (type 2) positive, Hes-1 positive, Integrinsubunits α6 and β1 positive, C-kit positive, MDR-1 positive, SST-R, 2,3, 4 positive, SUR-1 positive, Kir 6.2 positive and cytokeratin-19negative, CD34 negative, CD45 negative, CD133 negative, MHC class Inegative and MHC class II negative staining, or liver stem cells, suchas nestin-positive staining, can be used in various diagnostic,pathological, or investigative procedures to identify, localize, andquantitate stem cells in tissues from a patient or experimental animal.

[0121] Identification of Stem Cells in Pancreatic Islets

[0122] Previous investigators have focused on ductal epithelial cells ofpancreatic islets or exocrine tissue as a possible source of stem cellsfor the neogenesis of islet endocrine cells. Nestin is an intermediatefilament protein that was cloned by screening a cDNA library from E15rat embryos with a monoclonal antibody named R.401 (Hockfield & McKay,1985; Lendahl et al., 1990). Nestin was primarily found inneuroepithelial stem cells and is expressed in the developing centralnervous system. After maximum levels are reached in the rat embryo atE16, nestin expression declines to almost undetectable levels in adultcerebral cortex, coinciding with terminal differentiation of earlynestin-expressing progenitor cells (Lendahl et al., 1990). Nestin wasinitially found exclusively in stem cells of the embryonic developingbrain and skeletal muscle (Lendahl et al., 1990). Later studiesidentified nestin-positive neural stem cells in the subependymal layerof the adult mammalian forebrain (Morshead et al., 1994).Nestin-positive stem cells have been shown to be pluripotential evenwhen isolated from adult mice or rat brain. For example, nestin-positivestem cells can generate all three major classes of neural cells inculture: neurons, astrocytes, and oligodendrocytes (Reynolds & Weiss,1996). Nestin-positive neural stem cells respond to spinal cord injuryby proliferation and degeneration of migratory cells that differentiateinto astrocytes, participate in scar formation (Johansson et al., 1999)and restore hematopoietic cells of the bone marrow after infusion intoirradiated mice (Bjornson et al., 1999).

[0123] Characterization of Stem Cells

[0124] Stem cells according to the invention can be identified by theirexpression of nestin and/or GLP-1R, ABCG2, Oct3/4, latrophilin (type 2),Hes-1, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R, 2, 3, 4, SUR-1and Kir 6.2, by, for example, FACS, immunocytochemical staining, RT-PCR,Southern Northern and Western blot analysis, and other such techniquesof cellular identification as known to one skilled in the art.

[0125] Immunocytochemical staining, for example, is carried outaccording to the following method. Cryosections (6 μM) prepared frompancreata or liver, as well as cells, are fixed with 4% paraformaldehydein phosphate. Cells are first blocked with 3% normal donkey serum for 30min at room temperature and incubated with a primary antisera to theprotein of interest overnight at 4° C. The antisera is rinsed off withPBS and incubated with the appropriate fluorescently labeled secondaryantisera for 1 hour at room temperature. Slides are then washed with PBSand coverslipped with fluorescent mounting medium (Kirkegaard and PerryLabs, Gaithersburg, Md.). Fluorescence images are obtained using a ZeissEpifluorescence microscope equipped with an Optronics TEC-470 CCD camera(Optronics Engineering, Goleta, Calif.) interfaced with a PowerMac 7100installed with IP Lab Spectrum analysis software (Signal Analytics Corp,Vienna, Va.).

[0126] Antisera useful according to the invention include the following:mouse monoclonal antibodies to human cytokeratin 19 (clone K4.62, Sigma,St. Louis, Mo.), rabbit polyclonal antisera to rat nestin and to IDX-1(prepared by immunizations of rabbits with a purified GST-nestin fusionprotein or the last twelve amino acids of rat IDX-1, respectively)(McManus et al., 1999, J. Neurosci., 19:9004-9015), rabbit polyclonalantisera to GLP-1R (Heller et al., 1997, Diabetes 46: 7851) antisera toABCG2 (MAB4146, Chemicon (Temecula, Calif.)) antisera to integrin αG(SC-6597, Santa Cruz), antisera to integrin β1, (SC-6622, Santa Cruz),antisera to HES 1 (AB5702, Chemicon), antisera to CD45 (31252X, BDPharmingen (San Diego, Calif.)), antisera to CD34 (MS-363-PO, NeoMarkers(Freemont, Calif.)), antisera to MHC I (MS-557-PO, NeoMarkers), antiserato MHC II (MS-162-PO, NeoMarkers) antisera to MDR-1 (p170) (MS-660-PO,NeoMarkers), antisera to Oct 3/4 (SC-5279, Santa Cruz (SC, Calif.)),antisera to SUR-1(SC-5789, Santa Cruz) antisera to KIR 6.2 (SC-11227,Santa Cruz), antisera to ABC G2 (SC-18841, Santa Cruz) antisera to c-kit(SC-1493, Santa Cruz), antisera to SSTR 2 (SC-11606, Santa Cruz),antisera to SSTR 3 (SC-11610, Santa Cruz) antisera to SSTR 4 (SC-11619,Santa Cruz), guinea pig anti-insulin and anti-pancreatic polypeptideantisera, obtained from Linco, St. Charles, Mo., and mouse antiglucagonand rabbit antisomatostatin antisera, purchased from Sigma (St. Louis,Mo.) and DAKO (Carpinteria, Calif.), respectively, mouse anti-humangalanin (Peninsula Laboratories, Belmont, Calif.), collagen IV antisera(Caltag Laboratories, San Francisco, Calif.), mouse anti-rat MHC class Iserum (Seroteck), and antirat MHC class II serum. The inventioncontemplates that other antisera directed to such markers, is available,or will be developed. Such other antisera is considered to be within thescope of the invention.

[0127] RT-PCR and Southern blot analysis are performed according to thefollowing methods. Total cellular RNA prepared from rat or human isletsis reverse transcribed and amplified by PCR for about 35 cyclesdepending on the desired degree of amplification, as describedpreviously (Daniel, et al., 1998, Endocrinology, 139:3721-3729).Oligonucleotides used as primers or amplimers for the PCR and as probesfor subsequent Southern blot hybridization are: Rat nestin: Forward,5′gcggggcggtgcgtgactac3′; Reverse, 5′aggcaagggggaagagaaggatgt3′;Hybridization, 5′aagctgaagccgaatttccttgggataccagagga3′. Rat keratin 19:Forward, 5′acagccagtacttcaagacc3′; Reverse, 5′ctgtgtcagcacgcacgtta3′;Hybridization, 5′tggattccacaccaggcattgaccatgcca3′. Rat NCAM: Forward,5′cagcgttggagagtccaaat3′; Reverse, 5′ttaaactcctgtggggttgg3′;Hybridization, 5′aaaccagcagcggatctcagtggtgtggaacgatgat3′. Rat IDX-1Forward, 5′atcactggagcagggaagt3′ Reverse, 5′gctactacgtttcttatct3′Hybridization, 5′gcgtggaaaagccagtggg3′ Human nestin: Forward,5′agaggggaattcctggag3′; Reverse, 5′ctgaggaccaggactctcta3′;Hybridization, 5′tatgaacgggctggagcagtctgaggaaagt3′. Human keratin:Forward, 5′cttttcgcgcgcccagcatt3′; Reverse, 5′gatcttcctgtccctcgagc3′;Hybridization, 5′aaccatgaggaggaaatcagtacgctgagg3′. Human glucagon:Forward, 5′atctggactccaggcgtgcc3′; Reverse, 5′agcaatgaattccttggcag3′;Hybridization, 5′cacgatgaatttgagagacatgctgaaggg3′; Human E-CadherinForward, 5′agaacagcacgtacacagcc 3′ Reverse, 5′cctccgaagaaacagcaaga 3′Hybridization, 5′tctcccttcacagcagaactaacacacggg 3′ Human transthyretinForward, 5′gcagtcctgccatcaatgtg 3′ Reverse, 5′gttggctgtgaataccacct 3′Hybridization, 5′ctggagagctgcatgggctcacaactgagg 3′ Human PancreaticAmylase Forward, 5′gactttccagcagtcccata 3′ Reverse,5′gtttacttcctgcagggaac 3′ Hybridization,5′ttgcactggagaaggattacgtggcgttcta 3′ Human procarboxypeptidase Forward,5′tgaaggcgagaaggtgttcc 3′ Reverse, 5′ftcgagatacaggcagatat 3′Hybridization, 5′agttagacttttatgtcctgcctgtgctca 3′ Human SynaptophysinForward, 5′cttcaggctgcaccaagtgt 3′ Reverse, 5′gttgaccatagtcaggctgg 3′Hybridization, 5′gtcagatgtgaagatggccacagacccaga 3′ Human HepatocyteGrowth Forward, 5′gcatcaaatgtcagccctgg 3′ Factor (HGF) Reverse,5′caacgctgacatggaattcc 3′ Hybridization,5′tcgaggtctcatggatcatacagaatcagg 3′ Human cMET (HGF-receptor) Forward,5′caatgtgagatgtctccagc 3′ Reverse, 5′ccttgtagattgcaggcaga 3′Hybridization, 5′ggactcccatccagtgtctccagaagtgat 3′ Human XBP-1 Forward,5′gagtagcagctcagactgcc 3′ Reverse, 5′gtagacctctgggagctcct 3′Hybridization, 5′cgcagcactcagactacgtgcacctctgca 3′ Human Glut-2 Forward,5′gcagctgctcaactaatcac 3′ Reverse, 5′tcagcagcacaagtcccact 3′Hybridization, 5′acgggcattcttattagtcagattattggt 3′ Human InsulinForward, 5′aggcttcttctacaca3′ Reverse, 5′caggctgcctgcacca 3′Hybridization, 5′aggcagaggacctgca 3′

[0128] Other such sequences are possible and such sequences areconsidered to be within the scope of the art. The invention includesoligonucleotides used as primers or amplimers for the PCR and as probesfor Southern analysis for any of the markers selected from the groupconsisting of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R, 2, 3,4, SUR-1, Kir 6.2, CD34, CD45, CD133, MHC class I and MHC class II. As ageneral guide, primers are selected from two different exons andencompass at least one intronic sequence. In addition, an RT minuscontrol is run for most samples. PCR amplification is effectuated at 94°C. for 1 min followed by 94° C. for 10 secs, 58/56° C. for 10 secs, 72°C. for 1 min, 35 cycles, and 72° C. for 2 min. The annealing temperatureis 58° C. for rat nestin and 56° C. for the remaining primer pairs.

[0129] For RT-PCR of mRNA isolated from a mammal that is not rat orhuman, oligonucleotides that are specific for the amplified nucleic acidfrom the mammalian species being analyzed are prepared. The selectionand use of such primers is known to one skilled in the art.

[0130] For Southern hybridization oligonucleotide probes are labeledwith an appropriate radionuclide, such as γ³²P ATP, using conventionaltechniques. Radiolabeled probes are hybridized to PCR productstransferred to nylon membranes at 37° C. for one hour, then washed in1×SSC+0.5% SDS at 55° C. for 10-20 min or 0.5×SCC+0.5% SDS at 420 forthe human PCR products.

[0131] Nestin as a Marker of Pancreatic Stem Cells

[0132] The inventors have now unexpectedly discovered that the pancreasof adult mammals, including humans, contains cells that express nestin.Importantly, the distribution of nestin-positive cells in the pancreasdoes not correspond to the distribution of hormone-producing cells. Forexample, fluorescently labeled antibodies specifically reactive toinsulin or glucagon label the beta and alpha cells of the islets,respectively, whereas the inventors have observed that in mice, rats,and humans, fluorescently labeled nestin antibodies localize only tocertain cells of the ductular epithelium and not to alpha, beta, delta,and pancreatic peptide producing cells in pancreatic islets (FIG. 1).The inventors also observed that antibodies specific for collagen IV, amarker for vascular endothelial cells, galanin, a marker of nerveendings, and cytokeratin 19, a marker for ductal cells, do notcolocalize with nestin antibodies. Furthermore, the inventors have foundthat nestin-positive islet cells do not co-label with antibodies forinsulin, glucagon, somatostatin, or pancreatic polypeptide (FIG. 1).This suggests that these nestin-containing cells are not endocrinecells, ductal cells, neural cells, or vascular endothelial cells, butmay represent a truly distinct cell type within the islet that has notpreviously been described. The inventors have found nestin-positivecells in the islets, as well as localized regions of the pancreaticducts, and within centroacinar regions of the exocrine pancreas.

[0133] The expression of nestin mRNA in isolated islets was detectedusing RT-PCR with RNA from isolated rat islets (FIG. 2). The functionalproperties of nestin-positive pancreatic cells were investigated usingcell culture techniques and by the isolation of nestin-positive cellsfrom islets, both of which are described below.

[0134] The inventors have also discovered that the liver of ratscontains cells that express nestin (FIG. 13).

[0135] ATP-Binding Cassette (ABC) Multi-Drug Rresistance Transporters(ABCG2 (Brcp1) and MDR-) as Markers of Pancreatic Stem Cells

[0136] Human islet-derived NIPs contain a substantial subpopulation ofSP cells that co-express ABCG2, MDR1 and nestin: ABCG2 expressiondefines the side population (SP) phenotype of pluripotential stem cellsin the bone marrow, by virtue of excluding the dye Hoechst 33342. MDR-1renders SP cells the capacity to replicate. SP cells (CD34 low/negative)do not proliferate and therefore cannot be expanded in vitro. The forcedexpression of MDR-1 in CD34 low negative SP cells causes them toproliferate (Bunting et al., 2000, Blood 96:902). NIPs are also CD34low/negative. Therefore NIPs are now defined as SP cells by virtue oftheir expression of ABCG2 and unlike marrow-derived SP cells, NIPsexpress MDR-1 and therefore can proliferate making it feasible to expandNIP SP cells ex vivo for purposes of transplantation.

[0137] Oct3/4 as a Marker of Pancreatic Stem Cells

[0138] NIPs express Oct3/4. Oct3/4 is a transcription factor belongingto the family of Pou homeodomain proteins (Niwa et al., 2002, Mol. Cell.Biol. 22:1526; Niwa et al., 2000, Nat. Genet. 24:328; Niwa, 2001, CellStruct Funct 26:137; Shimazaki et al., 1993, EMBO 12:4489; Wang et al.,1996, Biochem Cell Biol 74:579; Nichols et al., 1998, Cell 95:379; .This is an important finding because the expression of Oct3/4 isstrictly restricted to stem/progenitor cells (Niwa et al. 2002, supra;Niwa et al., 2000, supra; Niwa, 2001, supra). NIPs robustly expressOct3/4, thereby defining them per se to be stem/progenitor cells. Thatis if Oct3/4 expression is absolutely restricted to stem/progenitorcells, and Oct3/4 is expressed in NIPs, then NIPs are stem/progenitorcells.

[0139] Integrin Subunits α6 and β1 as a Marker of Pancreatic Stem Cells

[0140] NIPs express the integrin subunits α and B1. α6/B1 integrin isthe laminin receptor (Giancotti and Ruoslahti, 1999, Science 285:1028).It has recently been shown that ES cells can be propagated in culturedishes coated with laminin and do not need to be grown on feeder celllayers (Xu et al., Nature Biotechnology, October 2001, 19:971). The cellsurface expressed integrins are essential for the maintenance of cellgrowth and development (see Giancotti et al., supra). The α6/B1 receptoris specific for recognition of laminin, which is an essential proteinexpressed in the blastocyst. Therefore α6/B1 is a marker of ES cells(stem/progenitor cells). NIPs express the integrin subunits α6 and B1.Therefore NIPs are similar to ES cells in this regard.

[0141] Hes-1 as a Marker of Pancreatic Stem Cells

[0142] The delta/Notch signaling pathway of lateral inhibition iscritical for embryonic development. Much as been written about theimportance of Notch signaling. Notch signaling is also important inNIPs. Notch signaling is absolutely required for the development of thepancreas (Apelquist et al., 1999, Nature 400:6747; Lammert et al., 2000,Mech Dev. 94:; Jensen et al., 2000, Nat. Genet., 24:36; Jansen et al.Diabetes 2000, 49:163). Ngn-3 is the key bHLH transcription factordeterminant required for the development of the endocrine pancreaslineage. Hes-1 is a powerful suppressor of Ngn-3 expression. NIPsrobustly express Hes-1. Therefore NIPs are progenitor cells preventedfrom differentiating into endocrine cells. Therefore a method to blockthe expression of Hes-1 in the NIPs will lead to their differentiationinto endocrine cells (β-cells). Such a method for blocking Hes-1expression could be the use of transfected antisense RNA, either byusing small interfering RNA oligonucleotides or an expression plasmidexpressing antisense Hes-1 RNA.

[0143] GLP-1R as a Marker of Pancreatic Stem Cells

[0144] The inventors have further discovered that the pancreas of adultmammals, including humans, contains cell that express the glucagon likepeptide-1 receptor (GLP-1R). The invention is further based on thediscovery that GLP-1R-positive cells can co-localize withnestin-positive cells. The invention is also based on the conversediscovery; that nestin-positive pancreatic stem cells can co-localizewith GLP-1R-positive cells. The glucagon gene encodes a multifunctionalproglucagon, that is differentially process by pro-hormone convertasesin the pancreas and intestine to yield glucagon and the glucagon-likepeptides (GLP). GLP-1 has been shown to bind specifically to a G-proteincoupled receptor found in pancreatic β-cells to stimulate insulinsecretion via a cAMP-dependent pathway (Kiefer and Habener, 1999,Endocr. Rev. 20: 876; Drucker, 1998 Diabetes, 47: 159). The presentinvention is based, in part, on the discovery that the nestin-positivestem cells found in the pancreatic islets and ducts, as described above,also are GLP-1R-positive.

[0145] Nestin-positive-Islet-Progenitor cells (NIPs) were isolated fromhuman islet tissue and reacted with rabbit polyclonal antisera to theGLP-1R (Heller et al., supra), following the general immunocytochemicalprocedures outlined above. Receptor immunoreactivity was detected ingreater than 60% of NIPs examined (FIG. 22A). To further confirm theimmunocytochemical identification of GLP-1R in NIPs, RT-PCR wasperformed on mRNA prepared from NIP cells. Amplification of NIP mRNAwith the amplification primers 5′ gtgtggcggccaattactac 3′ (Forward); 5′cttggcaagtctgcatttga 3′ (Reverse) produced the expected 346 bp product(FIG. 22B) indicating that, in addition to expressing the GLP-1Rprotein, NIPs have the biosynthetic ability to produce GLP-1R. GLP-1R,therefore, in addition to nestin, is useful in the present invention asa marker for pancreatic stem cells.

[0146] Cytokeratin-19 as a Marker for a Distinct Population of DuctEpithelial Cells

[0147] Cytokeratin-19 (CK-19) is another intermediate filament protein.CK-19 and related cytokeratins have previously been found to beexpressed in pancreatic ductal cells (Bouwens et al., 1994). Theinventors have discovered, however, that while CK-19 expression isindeed confined to the ductules, fluorescent antibodies specific forCK-19 label distinct ductal cells from those labeled withnestin-specific antibodies. This suggests that nestin-positive cells inislets may be a distinct cell type of ductal cell from CK-19 positivecells.

[0148] NIPs are CD34 Low/negative

[0149] The SP fraction of NIPs (left lower gate of FIG. 19B) express lowto null levels of CD34. This finding is important because CD34 lownegative marrow-derived SP cells, as contrasted to CD34-positive SPcells, are highly pluripotential (see Goodell, 1999, Blood, 94:12545).Marrow-derived CD34 low/negative SP cells have a limited, if any,capacity to proliferate (Goodell et al., 1996, J. Exp. Med., 183:1797;Goodell et al., 1997 Nature Medicine, 3:1337). CD34 low/negative NIPs doproliferate in vitro, because they express Mdr-1.

[0150] Additional Markers of Pancreatic Stem Cells

[0151] The invention also provides for pancreatic stem cells that arepositive for at least one of the markers selected from the groupconsisting of latrophilin (type 2) (Sudhof, 2001, Annu Rev Neurosci24:933), C-kit (CD117) (Gibson et al., 2002, Adv Anat Pathol 9:65),SSTR-2, 3 and 4 (somatostatin receptors) (Schulz et al., 2000, J PhysiolParis 94:259), SUR-1 (sulonylurea receptor) (Winarto et al., 2001, ArchHistol Cytol 64:59; Landgraf, 2000, Drugs Aging, 17:411), and Kir 6.2(inward rectifying K+ channel subunit with SUR-1 (Winarto et al., supra;Landgraf, supra).

[0152] The invention also provides for pancreatic stem cells that arenegative for at least one of CD45 (Sasaki et al., 2001, Int J BiochemCell Biol 33:1041), CD133 (Kobari et al., 2001, J Hematother Stem CellRes 10:273), MHC class II and MHC class II.

[0153] Isolated Stem Cells from Pancreatic Islets and Their Use

[0154] Stem cells can be isolated from a preparation of pancreatictissue, for example, islets obtained from a biopsy sample of tissue froma diabetic patient. The stem cells can then be expanded ex vivo and theresulting cells transplanted back into the donor as an isograft. Insidethe donor, they may differentiate to provide insulin-secreting cellssuch as beta cells to replace beta cells lost to the autoimmune attackwhich caused the diabetes. This approach can overcome the problems ofimmune rejection resulting from transplantation of tissue, for example,islets from another individual who might serve as the donor. In oneembodiment of the invention, the use of isografted stem cells allowsanother technique to be performed in an effort to avoid the immunerejection, namely genetic therapy of the transplanted cells to renderthem resistant to immune attack, such as the autoimmunity present inindividuals with type 1 diabetes. A further advantage of using stemcells over whole islets is that transplanted stem cells candifferentiate in situ and better adapt to the host environment, forexample, providing appropriate microcirculation and a complement ofdifferent islet cell types which responds to the physiological needs ofthe host. Another embodiment of the invention contemplates the use ofpartially differentiated stem cells ex vivo, for example, to formprogenitor cells, which are subsequently transplanted into the host,with further differentiation optionally taking place within the host.Although the use of an isograft of stem cells, progenitor cells, orpseudo-islets is preferred, another embodiment contemplates the use ofan allograft of stem cells, progenitor cells, or pseudo-islets obtainedfrom another individual or from a mammal of another species.

[0155] In yet another embodiment of the invention, the stem cells areimmunologically blind or immunoprivileged. In one embodiment of thisaspect of the invention, immunoprivileged stem cells do not expresssufficient amounts of class I and/or class II major histocompatibilityantigens (a.k.a. HLA or human leukocyte antigen) to elicit an immuneresponse from the host. For example, these stem cells, obtained fromallogeneic or xenogeneic sources do not initiate a host versus graftresponse in immunocompetent transplant recipients.

[0156] In another embodiment of this aspect of the invention,immunoprivileged stem cells do not express class I MHC antigens and/orclass II MHC antigens. These stem cells, obtained from allogeneic orxenogeneic sources do not initiate a host versus graft response inimmunocompetent transplant recipients.

[0157] In another embodiment of the invention, human tissue graftscomprising stem cells express both human specific class I and class IIMHC antigens, but are recognized by immunocompetent mice as self, and donot undergo host versus graft rejection. These stem cells, obtained fromallogeneic or xenogeneic sources do not initiate a host versus graftresponse in immunocompetent transplant recipients.

[0158] The invention also provides for methods of isolating stem cellsfrom a xenogenic donor, and transplanting the resulting cells into amammal of another species (e.g. murine stem cells are transplanted intoa human, for example, a diabetic human patient) as a xenograft.

[0159] The invention provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of nestin-positive stem cellswherein the stem cells are cultured for a period of time, for example,2-4 hours, 4-5 hours, 5-10 hours or 1-3 days prior to transplantation.The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of a stem cell that is positive forat least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2,SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one or all ofCD34, CD45, CD133, MHC class I and MHC class II wherein the stem cellsare cultured for a period of time, for example, 2-4 hours, 4-5 hours,5-10 hours or 1-3 days prior to transplantation.

[0160] The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of nestin-positive stem cellswherein the stem cell is expanded for a period of time, for example, 2-4hours, 4-5 hours, 5-10 hours or 1-3 days prior to transplantation togive rise by cell division to other stem cells or progenitor cells.

[0161] The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of a stem cell that is positive forat least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2,SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one or all ofCD34, CD45, CD133, MHC class I and MHC class II wherein the stem cell isexpanded for a period of time, for example, 2-4 hours, 4-5 hours, 5-10hours or 1-3 days prior to transplantation.

[0162] The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of stem cells wherein thenestin-positive stem cells are induced to differentiate into aprogenitor cell by treatment with an agent selected from the groupconsisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, a nucleicacid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin, activin A,TGF-β, and combinations thereof for a period of time, for example, 2-4hours, 4-5 hours, 5-10 hours or 1-3 days prior to transplantation.

[0163] In the case of a pancreatic stem cell, the stem cell subsequentlydifferentiates into a pancreatic progenitor cell.

[0164] The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants of a stem cell that is positive forat least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2,SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one or all ofCD34, CD45, CD133, MHC class I and MHC class II wherein the stem cellsare induced to differentiate into a progenitor cell by treatment with anagent selected from the group consisting of EGF, bFGF-2, high glucose,KGF, HGF/SF, IDX-1, a nucleic acid molecule encoding IDX-1, GLP-1,exendin-4, betacellulin, activin A, TGF-β, and combinations thereof fora period of time, for example, 2-4 hours, 4-5 hours, 5-10 hours or 1-3days prior to transplantation. In the case of a pancreatic stem cell,the stem cell subsequently differentiates into a pancreatic progenitorcell.

[0165] The invention provides for methods of performing isogeneic,allogeneic or xenogeneic transplants wherein nestin-positive stem cellsare not cultured, expanded or differentiated prior to transplantation orwherein nestin-positive stem cells are cultured and/or expanded and/ordifferentiated prior to transplantation.

[0166] The invention also provides for methods of performing isogeneic,allogeneic or xenogeneic transplants wherein a stem cell that ispositive for at least one of ABCG2, Oct3/4, GLP-1 receptor, latrophilin(type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1,SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not express one orall of CD34, CD45, CD133, MHC class I and MHC class II are not cultured,expanded or differentiated prior to transplantation or wherein a stemcell that is positive for at least one of ABCG2, Oct3/4, GLP-1 receptor,latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 and β1, C-kit,MDR-1, SST-R2, SST-R3, SST-R4, SUR-1, Kir 6.2 and/or does not expressone or all of CD34, CD45, CD133, MHC class I and MHC class II iscultured and/or expanded and/or differentiated prior to transplantation.

[0167] Nestin-positive cells can be proliferated in culture fromisolated pancreatic islets and subsequently isolated to form a stem cellline capable of essentially unlimited propagation.

[0168] In another embodiment, a stem cell that is positive for at leastone of ABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1,Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SST-R2, SST-R3,SST-R4, SUR-1, Kir 6.2 and/or does not express one or all of CD34, CD45,CD133, MHC class I and MHC class II can be proliferated in culture fromisolated pancreatic islets and subsequently isolated to form a stem cellline capable of essentially unlimited propagation.

[0169] The inventors discovered that nestin expressing cells grow out ofcultured islets and can be observed growing around the islets as earlyas about four days in culture. These cells have a neuron-like morphology(FIG. 3), show nestin-positive staining, and express nestin mRNA. Isletscontaining nestin-positive cells can be separated from other cells,e.g., fibroblasts, that proliferate from the cultured islets by exposinga suspension of the islets to concanavalin A. The islets containingnestin-positive stem cells will not adhere to a concanavalin A coatedculture vessel, for example, allowing the islets to be simply decantedwhile other cell types remain attached to the vessel. The islets arethen plated on wells that do not have a concanavalin A coating, wherethey adhere. The details of the culture and isolation procedure aredescribed for rat cells in Example 1 below. Similar results have beenobtained with human cells.

[0170] Formation of Pseudo-Islets and Ductal Structures in Culture

[0171] One embodiment of the invention provides an alternative totransplantation of stem cells or progenitor cells by causing them toform pseudo-islet like aggregates that can be transplanted into apatient with insufficient islet cell mass to maintain physiologicalcontrol without hormone therapy. Islet-derived stem cells can beprepared from cultured islets as indicated above or obtained from apropagating stem cell line. The stem cells can then be induced todifferentiate by exposing them to various growth factors. This processis illustrated in Examples 2 and 3.

[0172] Differentiation of Stem Cells or Progenitor Cells to Islet Cells

[0173] Growth factors that may induce differentiation of pancreatic stemcells include but are not limited to EGF-2, basic FGF, high glucose,KGF, HGF/SF, GLP-1, exendin-4, betacellulin, activin A, TGF-β, andcombinations thereof. GLP-1 refers to glucagon-like peptide-1. Highglucose refers to a higher glucose concentration than the concentrationnormally used in culturing the stem cells. For example, the stem cellscan be normally cultured and propagated in about 5.6 mM glucose, and thehigh glucose concentration refers to another concentration above 5.6 mM.In the preferred embodiment, a concentration of 16.7 mM is contemplated.In Example 2, one possible growth factor treatment is described usingbasic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).

[0174] In addition to growth factors added to the medium of culturedcells, further growth factors can contribute to differentiation whenstem cells are implanted into an animal or a human. In that situation,many growth factors which are either known or unknown may be secreted byendogenous cells and exposed to the stem cells in situ. Implanted stemcells can be induced to differentiate by any combination of endogenousand exogenously administered growth factors that are compatible with thedesired outcome, i.e., the final differentiated cell type or types andthe anatomical structures (e.g., tissues or organs) formed.

[0175] One embodiment provides an approach to stimulatingdifferentiation, that is to administer downstream effectors of growthfactors or to transfect stem cells or progenitor cells with a nucleicacid molecule encoding such effectors. One example is IDX-1, which is atranscription factor induced by GLP-1 or exendin-4. Introducingeffectors such as IDX-1 can trigger differentiation to form endocrineislet cells.

[0176] Analysis of Graft Rejection

[0177] The invention provides for an in vivo procedure for evaluatingthe survival of transplanted material. Experimental transplant rejectionis analyzed by transplanting an immunosuppressed or anon-immunosuppressed mammal, with a stem cell or a pseudo-islet likeaggregate according to the invention.

[0178] For example, non-immunosuppressed C57BL/6 mice are transplanted(for example, under the renal capsules) with human stem cells accordingto the invention. Graft rejection is analyzed by sacrificing thetransplant recipient and staining for viability, or performingimmunocytochemical staining at the site of the grafted material (i.e.,an organ or tissue present at the site of the grafted material) at asuitable post-transplantation time point. The time point at whichstaining (for example hematoxylin/eosin or Immunostaining) of the siteof the grafted material is made can vary, for example, according to theaverage survival time, or the expected survival time of a transplantedmammal. The site of the graft is analyzed, for example by staining, 1day to 10 years (i.e., 1, 5, 10, 30, 100 or more days, 1, 2, 5, or 10years) post-transplantation, preferably 10 days to 1 yearpost-transplantation and most preferably, 10-100 dayspost-transplantation. For example, if transplanted material isintroduced under the renal capsule of a mouse, the kidney of thetransplanted mouse is inspected. Transplanted material is successfullyengrafted (i.e., not rejected) if, the transplanted material is stilldetectable and/or the transplanted material has proliferated into atissue mass.

[0179] Detection of the transplanted material and proliferation of thetransplanted material is determined, for example, by hematoxylin/eosinstaining of a frozen section prepared from the transplant site (i.e.,the kidney) and the detection of new growth that is not derived from thetransplant recipient (i.e., not host kidney derived). In the case ofxenogeneic transplantation, transplanted material is successfullyengrafted if specific immunostaining with antisera specific for anantigen from the species from which the transplanted material isderived, according to methods of immunocytochemical staining known inthe art and described herein, identifies positive cells. Alternatively,in embodiments wherein a xenogeneic transplantation is performed,transplanted material is successfully engrafted if molecules (i.e., aprotein or an antigen) derived from the transplant species (that is thespecies from which the transplanted material is derived) are detected inthe blood of the transplant recipient.

[0180] As used herein, “rejection” refers to rejection of transplantedmaterial by the immune system of the host. in one embodiment, “rejectionmeans an occurrence of more than 90% cell or tissue necrosis of thetransplanted material in response to the immune response of the host. Inanother embodiment, “rejection” means a decrease in the viability suchthat the viability of the transplanted material is decreased by 90% ormore as compared to the viability of the transplanted material prior totransplantation, in response to the immune response of the host. Adecrease in viability can be determined by methods well known in theart, including but not limited to trypan blue exclusion staining. Inanother embodiment, “rejection” means failure of the transplantedmaterial to proliferate. Proliferation can be measured by methods knownin the art including but not limited to hematoxylin/eosin staining. Theoccurrence of transplant rejection and/or the speed at which rejectionoccurs following transplantation will vary depending on factors,including but not limited to the transplanted material (i.e., the celltype, or the cell number) or the host (i.e., whether or not the host isimmunotolerant and/or has been treated with an immunosuppressive agent.

[0181] Methods of Transplantation

[0182] The invention provides for methods of transplantation in to amammal. A stem cell, progenitor cell, or differentiated cell is“transplanted” or “introduced” into a mammal when it is transferred froma culture vessel into a patient.

[0183] Transplantation, according to the invention can include the stepsof isolating a stem cell according to the invention and transferring thestem cell into a mammal or a patient. Transplantation according to theinvention can involve transferring a stem cell into a mammal or apatient by injection of a cell suspension into the mammal or patient,surgical implantation of a cell mass into a tissue or organ of themammal or patient, or perfusion of a tissue or organ with a cellsuspension. The route of transferring the stem cell or transplantation,will be determined by the need for the cell to reside in a particulartissue or organ and by the ability of the cell to find and be retainedby the desired target tissue or organ. In the case where a transplantedcell is to reside in a particular location, it can be surgically placedinto a tissue or organ or simply injected into the bloodstream if thecell has the capability to migrate to the desired target organ.Transplantation, according to the invention, can include the steps ofisolating a stem cell according to the invention, and culturing andtransferring the stem cell into a mammal or a patient. In anotherembodiment, transplantation, as used herein, can include the steps ofisolating a stem cell according to the invention, differentiating thestem cell, and transferring the stem cell into a mammal or a patient.Transplantation, as used herein, can include the steps of isolating astem cell according to the invention, differentiating and expanding thestem cell and transferring the stem cell into a mammal or a patient.

[0184] Methods of Treating Insulin-Dependent Diabetes Using PancreaticStem Cells

[0185] Stem cells are useful to replace lost beta cells from Type 1diabetes patients or to increase the overall numbers of beta cells inType 2 diabetes patients. The diabetes patient will preferably serve asthe donor of pancreatic tissue used to produce stem cells, progenitorcells, or pseudo-islet like aggregates. Stem cells exist within theadult pancreatic islets as well as the pancreatic ducts. After adiabetic patient undergoes pancreatic biopsy, islets are isolated fromthe biopsy tissue and prepared for culture ex vivo preferably within 24hours. Stem cells can be proliferated and isolated by the methodsdescribed above within 2-3 weeks. Stem cells can be transplanted backinto the patient directly following isolation or after a period ofdifferentiation which is induced by growth factors. Islets can beproduced by subculture as described in Example 2. The whole process ofsurgical pancreas biopsy and transplantation can be performed within aperiod of about 30 days.

[0186] In one embodiment of the invention, pluripotential stem cells areused. These cells are immunologically blinded or immunoloprivileged,such that in allogeneic or xenogeneic transplants, they are recognizedas self by the recipient, and are not MHC restricted by class I or classII antigens. In one aspect of this embodiment of the invention, thesecells do not express MHC class I and/or class II antigens.

[0187] In another embodiment of the invention, the recipient of thetransplant may demonstrate host vs. graft rejection of othertransplanted cells, which can be combated by the administration ofblocking antibodies to, for example, an autoantigen such as GAD65, bythe administration of one or more immunosuppressive drugs describedherein, or by any method known in the art to prevent or ameliorateautoimmune rejection.

[0188] Alternatively, stem cells isolated from a non-human mammalaccording to the invention, are transplanted into a human diabetespatient. Prior to the transplantation step the stem cells may becultured, and/or expanded and/or differentiated.

[0189] Methods of Treating Patients Suffering from Liver Disease UsingPancreatic Stem Cells

[0190] The ability of pancreatic stem or progenitor cells totransdifferentiate to form hepatocytes is well known (Bisgaard &Thorgeirsson, 1991). The pancreatic stem cells of the present inventioncan be used to provide hepatocytes for a patient suffering from a liverdisease such as cirrosis, hepatitis, or hepatic cancer in which thefunctional mass of hepatic tissue has been reduced. The stem cells ofthe invention can also be treated by gene therapy to correct a geneticdefect and introduced into a patient to restore hepatic function.Nestin-positive stem cells can be differentiated either in culture or invivo by applying one or more growth factors, or other treatment such astransfection with a nucleic acid molecule, that results indifferentiation of the stem cells to hepatocytes. In one embodiment, theinvention contemplates the use of cyclopamine to suppress, for example,sonic hedgehog, resulting in hepatocyte formation. In another embodimentof the invention, the stem cells can be transplanted without any ex vivotreatment and the appropriate growth factors can be provided in situwithin the patient's body. In yet another embodiment, the stem calls canbe treated with growth factors or other agents ex vivo and subsequentlytransplanted into the patient in a partially differentiated orterminally differentiated state. Other aspects of the invention,including methods of transfecting stem cells or progenitor cells,dosages and routes of administration, pharmaceutical compositions,donor-isograft protocols, and immunosuppression methods, can bepracticed with transdifferentiation to hepatocytes just as for thedifferentiation to pancreatic tissues.

[0191] The invention specifically contemplates transplanting intopatients isogeneic, allogeneic, or xenogeneic stem cells, or anycombination thereof.

[0192] Methods of Stem Cell Transfection

[0193] A variety of methods are available for gene transfer intopancreatic stem cells. Calcium phosphate precipitated DNA has been usedbut provides a low efficiency of transformation, especially fornonadherent cells. In addition, calcium phosphate precipitated DNAmethods often result in insertion of multiple tandem repeats, increasingthe likelihood of disrupting gene function of either endogenous orexogenous DNA (Boggs, 1990). The use of cationic lipids, e.g., in theform of liposomes, is also an effective method of packaging DNA fortransfecting eukaryotic cells, and several commercial preparations ofcationic lipids are available. Electroporation provides improvedtransformation efficiency over the calcium phosphate protocol. It hasthe advantage of providing a single copy insert at a single site in thegenome. Direct microinjection of DNA into the nucleus of cells is yetanother method of gene transfer. It has been shown to provideefficiencies of nearly 100% for short-term transfection, and 20% forstable DNA integration. Microinjection bypasses the sometimesproblematic cellular transport of exogenous DNA through the cytoplasm.The protocol requires small volumes of materials. It allows for theintroduction of known amounts of DNA per cell. The ability to obtain avirtually pure population of stem cells would improve the feasibility ofthe microinjection approach to targeted gene modification of pancreaticstem cells. Microinjection is a tedious, highly specialized protocol,however. The very nature of the protocol limits the number of cells thatcan be injected at any given time, making its use in large scaleproduction limited. Gene insertion into pancreatic stem cells usingretroviral methods is the preferred method. Retroviruses provide arandom, single-copy, single-site insert at very high transfectionefficiencies. Other such transfection methods are known to one skilledin the art and are considered to be within the scope of this invention.

[0194] Retroviral Transformation of Pancreatic Stem Cells

[0195] Gene transfer protocols for pancreatic cells can involveretroviral vectors as the “helper virus” (i.e., encapsidation-defectiveviral genomes which carry the foreign gene of interest but is unable toform complete viral particles). Other carriers such as DNA-mediatedtransfer, adenovirus, SV40, adeno-associated virus, and herpes simplexvirus vectors can also be employed. Several factors should be consideredwhen selecting the appropriate vector for infection. It is sometimespreferable to use a viral long terminal repeat or a strong internalpromoter to express the foreign gene rather than rely on splicedsubgenomic RNA.

[0196] The two primary methods of stem cell transformation areco-culture and supernatant infection. Supernatant infection involvesrepeated exposure of stem cells to the viral supernatant. Co-cultureinvolves the commingling of stem cells and an infected “package cellline” (see below) for periods of 24 to 48 hours. Co-culture is typicallymore efficient than supernatant infection for stem cell transformation.After co-culture, infected stem cells are often further cultured toestablish a long term culture (LTC).

[0197] The cell line containing the helper virus is referred to as thepackage cell line. A variety of package cell lines are currentlyavailable. An important feature of the package cell line is that it doesnot produce replication-competent helper virus.

[0198] In one embodiment of the invention animals or patients from whomstem cells are harvested may be treated with 5- fluorouracil (5-FU)prior to extraction. 5-FU treated stem cells are more susceptible toretroviral infection than untreated cells. 5-FU stem cells dramaticallyreduce the number of clonogenic progenitors, however.

[0199] In another embodiment, harvested stem cells may be exposed tovarious growth factors, such as those employed to promote proliferationor differentiation of pancreatic stem cells. Growth factors can beintroduced in culture before, during, or after infection to improve cellreplication and transduction. Studies report the use of growth factorsincrease transformation efficiency from 30 to 80%.

[0200] Typical Retroviral Transformation Protocol

[0201] The ex vivo transduction of mammalian pancreatic stem cells andsubsequent transplantation into nonablated recipients sufficient toobtain significant engraftment and gene expression in various tissuescontaining their progeny cells has been shown in mice. The target cellsare cultured for 2-4 days in the presence of a suitable vectorcontaining the gene of interest, before being injected in to therecipient.

[0202] Specifically, bone marrow stem cells were harvested from maledonor (4-8 weeks old) BALB/c AnNCr mice (National Cancer Institute,Division of Cancer Treatment Animal Program, Frederick, Md.). The cellswere plated at a density of 1-2×10⁷ cells/10 cm dish and cultured for 48hours in Dulbecco's modified Eagle's medium (DMEM) containing; 10%heat-inactivated fetal bovine serum, glutamine, Pen/Strep, 100 U/ml ofinterleukin-6 (IL-6) and stem cell factor (SCF; Immunex, Seattle, Wash.)to stimulate cell growth (Schiffmann, et. al., 1995).

[0203] Concurrently, a viral package cell line was cultured for 24hours. The package cell line used by Schiffmann, et al. was GP+E86 andthe viral vector was the LG retroviral vector based on the LN series ofretroviral vectors.

[0204] After the appropriate incubation period, 1-2×10⁷ stem cells wereplated on a 10 cm dish containing the viral package cells andco-cultured for 48 hours in the presence of 8 μg/ml of polybrene andunder the same growth factor stimulation conditions as the donor stemcells. The stem cells were then harvested, washed of growth media andinjected into recipient mice at dosages of 2×10⁷ cells/injection formultiple injections (total of 5 injections either daily or weekly).

[0205] Successful stem cell transduction and engraftment of stem cellscan be determined through, for example, PCR analysis, immunocytochemicalstaining, Southern Northern or Western blotting, or by other suchtechniques known to one skilled in the art.

[0206] Mammals

[0207] Mammals that are useful according to the invention include anymammal (for example, human, mouse, rat, sheep, rabbit, goat, monkey,horse, hamster, pig or cow). A non-human mammal according to theinvention is any mammal that is not a human, including but not limitedto a mouse, rat, sheep, rabbit, goat, monkey, horse, hamster, pig or acow.

[0208] Dosage and Mode of Administration

[0209] By way of example, a patient in need of pancreatic stem cells asdescribed herein can be treated as follows. Cells of the invention canbe administered to the patient, preferably in a biologically compatiblesolution or a pharmaceutically acceptable delivery vehicle, byingestion, injection, inhalation or any number of other methods. Apreferred method is endoscopic retrograde injection. Another preferredmethod is injection into the pancreatic artery. Another preferred methodis injection or placement of the cells or pseudo-islet like aggregatesinto the space under the renal capsule. The dosages administered willvary from patient to patient; a “therapeutically effective dose” can bedetermined, for example but not limited to, by the level of enhancementof function (e.g., insulin production or plasma glucose levels).Monitoring levels of stem cell introduction, the level of expression ofcertain genes affected by such transfer, and/or the presence or levelsof the encoded product will also enable one skilled in the art to selectand adjust the dosages administered. Generally, a composition includingstem cells will be administered in a single dose in the range of 10⁵-10⁸cells per kg body weight, preferably in the range of 10⁶-10⁷ cells perkg body weight. This dosage may be repeated daily, weekly, monthly,yearly, or as considered appropriate by the treating physician. Theinvention provides that cell populations can also be removed from thepatient or otherwise provided, expanded ex vivo, transduced with aplasmid containing a therapeutic gene if desired, and then reintroducedinto the patient.

[0210] Pharmaceutical Compositions

[0211] The invention provides for compositions comprising a stem cellaccording to the invention admixed with a physiologically compatiblecarrier. As used herein, “physiologically compatible carrier” refers toa physiologically acceptable diluent such as water, phosphate bufferedsaline, or saline, and further may include an adjuvant. Adjuvants suchas incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide,or alum are materials well known in the art.

[0212] The invention also provides for pharmaceutical compositions. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carrier preparationswhich can be used pharmaceutically.

[0213] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for ingestion by the patient.

[0214] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethyl cellulose; and gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0215] Dragee cores are provided with suitable coatings such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

[0216] Pharmaceutical preparations which can be used orally includepush-fit capsules, made of gelatin, as well as soft, sealed capsulesmade of gelatin and a coating such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binderssuch as lactose or starches, lubricants such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

[0217] Pharmaceutical formulations for parenteral administration includeaqueous solutions of active compounds. For injection, the pharmaceuticalcompositions of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer' solution, or physiologically buffered saline. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Additionally, suspensions of the active solventsor vehicles include fatty oils such as sesame oil, or synthetic fattyacid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0218] For nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0219] The pharmaceutical compositions of the present invention may bemanufactured in a manner known in the art, e.g. by means of conventionalmixing, dissolving, granulating, dragee-making, levitating, emulsifying,encapsulating, entrapping or lyophilizing processes.

[0220] The pharmaceutical composition maybe provided as a salt and canbe formed with many acids, including but not limited to hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. . . Salts tendto be more soluble in aqueous or other protonic solvents that are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a Ph range of 4.5 to 5.5 that is combined with bufferprior to use.

[0221] After pharmaceutical compositions comprising a compound of theinvention formulated in a acceptable carrier have been prepared, theycan be placed in an appropriate container and labeled for treatment ofan indicated condition with information including amount, frequency andmethod of administration.

[0222] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific examples, which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLE 1

[0223] Isolation of Nestin-Positive Stem Cells from Rat Pancreas

[0224] Rat islets were isolated from the pancreata of 2-3 month oldSprague-Dawley rats using the collagenase digestion method described byLacy and Kostianovsky. Human islets were provided by the DiabetesResearch Institute, Miami, Fla. using collagenase digestion. The isletswere cultured for 96 hrs at 37° C. in 12-well plates (Falcon 3043plates, Becton Dickinson, Lincoln Park, N.J.) that had been coated withconcanavalin A. The culture medium was RPMI 1640 supplemented with 10%fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES buffer, 100 μg/mlstreptomycin, 100 units/ml penicillin, 0.25 μg/ml amphotericin B (GIBCOBRL, Life Science Technology, Gaithersburg, Md.), and 71.5 mMβ-mercaptoethanol (Sigma, St. Louis, Mo.).

[0225] After 96 hrs, fibroblasts and other non-islet cells had adheredto the surface of concanavalin A coated wells and the islets remainedfloating (did not adhere to the surface). At this time, the mediacontaining the islets were removed, centrifuged down, and the purgedislets replated in 12-well plates without a coating of concanavalin A.The islets were then cultured in the above RPMI 1640 medium supplementedwith 20 ng/ml of basic fibroblast growth factor-2 and 20 ng/ml ofepidermal growth factor.

[0226] The islets adhered to the surface of the plates, and cells grewout and away from the islets in a monolayer. These cells that form amonolayer were nestin-positive by immunostaining with a rabbit anti-ratnestin antiserum developed by Dr. Mario Vallejo at the MassachusettsGeneral Hospital. Other nestin antibodies may be used, for example theR.401 antibody described hereinabove, or the MAB533 antibody. Amonoclonal antibody specific for rat embryo spinal cord nestin, MAB353,ATCC No. 1023889; is described in Journal of Neuroscience 1996;16:1901-100; and also available from Chemicon International, Single OakDr., Temecula, Calif. 92590 USA. After two weeks of culture, several(3-5) of the nestin-positive monolayer cells were removed by pickingwith a capillary tube (cylinder cloning) and were replated on the12-well plates (not coated with concanavalin A) and cultured in the RPMI1640 medium further supplemented with bFGF-2 and EGF. The cellspropagated at a rapid rate and reached confluence after six days ofculture. After 12 days of culture, the cell monolayer formed waves inwhich they begin to pile up in a co-linear manner. On day 15 of culture,the cell waves began to condense, migrate into spheroid bodies and byday 17 the surface of the wells contained these spheroid bodies (ca. 100μm in diameter), empty spaces, and a few areas of remaining monolayercells. Several of these monolayer cells were re-picked and re-cloned andthe process described above occurred again in precisely the sametemporal sequence.

EXAMPLE 2

[0227] Differentiation of Pancreatic Stem Cells to Form Islet

[0228] Pancreatic islets from rats were first cultured in RPMI mediumcontaining 10% fetal bovine serum using concanavalin-A coated 12-wellplates. The islets were maintained in culture for three days in theabsence of added growth factors other than those supplied by fetalbovine serum. After this period, during which the islets did not attach,the islets were transferred to fresh plates without concanavalin A. Thestein cells were then stimulated to proliferate out from the islets as amonolayer by exposing them to bFGF-2 (20 ng/ml) and EGF (20 ng/ml) for24 days. After the 24 day period, the monolayer was confluent. Amongthem was a population of cells surrounding the islets. Cells from thatpopulation were picked and subcloned into new 12-well plates and againcultured in the medium containing bFGF and EGF. The subcloned cellsproliferated rapidly into a monolayer in a clonal fashion, expandingfrom the center to the periphery. The cells became confluent at day 6and then started to form a wave of overlapping cells on day 12. By day17 the cells migrated almost entirely into spherical structures andtubular structures resembling islet-like structures (pseudo-islet likeaggregates) and duct-like structures (pseudo-ducts) (FIG. 4). RT-PCRanalysis revealed that the pseudo-islet like aggregates were expressingNCAM (a marker for endocrine cells, see FIG. 5), cytokeratin 19 (amarker for ductal cells, see FIG. 5), and the transcription factorbrain-4 (a beta cell marker). Treatment with growth factors is requiredto achieve terminal differentiation to mature islet cells.

EXAMPLE 3

[0229] Isolation and Culture of Human or Rat Pancreatic Islets

[0230] Human pancreatic islets were isolated and cultured. Human islettissue was obtained from the islet distribution program of the CellTransplant Center, Diabetes Research Institute, University of MiamiSchool of Medicine and the Juvenile Diabetes Foundation Center for IsletTransplantation, Harvard Medical School, Boston, Mass. Thoroughly washedislets were handpicked, suspended in modified RPMI 1640 media (11.1 mMglucose) supplemented with 10% fetal bovine serum, 10 mM HEPES buffer, 1mM sodium pyruvate, 100 U per mL penicillin G sodium, 100 μg per mLstreptomycin sulfate, 0.25 ng per mL amphotericin B, and 71.5 μMβ-mercaptoethanol, and added to Falcon 3043 12-well tissue cultureplates that had been coated with Concanavalin A (ConA). The isletpreparation was incubated for 96 hrs at 37° C. with 95% air and 5% CO₂.In these conditions, many islets remained in suspension (floated),whereas fibroblasts and other non-islet cells attached to thesubstratum. After 96 h of incubation the media containing the suspendedislets was carefully removed, the islets were manually picked andresuspended in the modified RPMI 1640 media now further supplementedwith 20 ng/mL each of basic fibroblast growth factor (bFGF) andepidermal growth factor (EGF). The islet suspension (containing 20-30islets per well) was added to 12-well tissue culture plates not coatedwith ConA. The islets immediately adhered to the surfaces of the plates.Within several days, a monolayer of cells was observed growing out andaway from the islets. In certain instances, human-derived cells werecultured in modified RPMI media containing 2.5 mM glucose, and severalgrowth factor combinations that include activin-A (2 nM), hepatocytegrowth factor (100 pM), or betacellulin (500 pM). In those experimentsin which cells were challenged with 10 mM nicotinamide, the mediacontained no serum or growth factors.

EXAMPLE 4

[0231] Effects of Glucose and GLP-1 on Differentiation of PancreaticStem Cells

[0232] Elevation of plasma glucose concentration leads to increasedpancreatic islet size. The effect of the glucose concentration in theculture medium was therefore investigated using isolated islets, whichcontain nestin-positive stem cells. Rat pancreatic islets were culturedin a medium containing high (16.7 mM) glucose or in normal (5.6 mM)glucose. After four days, RT-PCR was performed to determine the level ofnestin mRNA. The results indicated a three-fold stimulation of nestinmRNA levels in the islets cultured in high glucose compared to theislets cultured in normal glucose (FIG. 6).

[0233] Similarly, injection of glucagon-like peptide-1 (GLP-1) into micewas found to increase islet mass by 2-fold in 48 hours. Knockout micehaving a disrupted gene for GLP-1 receptor were examined for nestinexpression in pancreatic islets. Immunostaining using a nexin antibodywas found to be markedly reduced compared to normal mice with GLP-1receptors.

[0234] Animal Model of Diabetes Mellitus

[0235] Treatments for diabetes mellitus type that result in relief ofits symptoms are tested in an animal which exhibits symptoms ofdiabetes. It is contemplated that the animal will serve as a model foragents and procedures useful in treating diabetes in humans. Potentialtreatments for diabetes can therefore be first examined in the animalmodel by administering the potential treatment to the animal andobserving the effects, and comparing the treated animals to untreatedcontrols.

[0236] The non-obese diabetic (NOD) mouse is an important model of typeI or insulin dependent diabetes mellitus and is a particularly relevantmodel for human diabetes (see Kikutano and Makino, 1992, Adv. Immunol.52:285 and references cited therein, herein incorporated by reference).The development of type I diabetes in NOD mice occurs spontaneously andsuddenly without any external stimuli. As NOD mice develop diabetes,they undergo a progressive destruction of β-cells which is caused by achronic autoimmune disease. The development of insulin-dependentdiabetes mellitus in NOD mice can be divided roughly into two phases:initiation of autoimmune insulitis (lymphocytic inflammation in thepancreatic islets) and promotion of islet destruction and overtdiabetes. Diabetic NOD mice begin life with euglycemia, or normal bloodglucose levels, but by about 15 to 16 weeks of age the NOD mice startbecoming hyperglycemic, indicating the destruction of the majority oftheir pancreatic β-cells and the corresponding inability of the pancreasto produce sufficient insulin. In addition to insulin deficiency andhyperglycemia, diabetic NOD mice experience severe glycosuria,polydypsia, and polyuria, accompanied by a rapid weight loss. Thus, boththe cause and the progression of the disease are similar to humanpatients afflicted with insulin dependent diabetes mellitus. Spontaneousremission is rarely observed in NOD mice, and these diabetic animals diewithin 1 to 2 months after the onset of diabetes unless they receiveinsulin therapy.

[0237] The NOD mouse is used as an animal model to test theeffectiveness of the various methods of treatment of diabetes byadministering a stem cell preparation according to the invention. Assuch, treatment via administration of stem cells are tested in the NODmouse for their effect on type I diabetes.

[0238] The stem cells are administered to a NOD mouse, typicallyintraperitoneally, according to the following dosage amounts. NOD miceare administered about 1×10¹ to 1×10 ⁴ cells per mouse. Administrationof the cells is started in the NOD mice at about 4 weeks of age, and iscontinued for 8 to 10 weeks, e.g., 3 times a week. The mice aremonitored for diabetes beginning at about 13 weeks of age, being testedtwice per week according to the methods described below. The effects oftreatment are determined by comparison of treated and untreated NODmice.

[0239] The effectiveness of the treatment methods of the invention ondiabetes in the NOD mice is monitored by assaying for diabetes in theNOD mice by means known to those of skill in the art, for example,examining the NOD mice for polydipsia, polyuria, glycosuria,hyperglycemia, and insulin deficiency, or weight loss. For instance, thelevel of urine glucose (glycosuria) can be monitored with Testape (EliLilly, Indianapolis, Ind.) and plasma glucose levels can be monitoredwith a Glucometer 3 Blood Glucose Meter (Miles, Inc., Elkhart, Ind.) asdescribed by Burkly, 1999, U.S. Pat. No. 5,888,507, herein incorporatedby reference. Monitoring urine glucose and plasma glucose levels bythese methods, NOD mice are considered diabetic after two consecutiveurine positive tests gave Testape values of +1 or higher or plasmaglucose levels >250 mg/dL (Burkly, 1999, supra). Another means ofassaying diabetes in NOD mice is to examine pancreatic insulin levels inNOD mice. For example, pancreatic insulin levels can be examined byimmunoassay and compared among treated and control mice (Yoon, U.S. Pat.No. 5,470,873, herein incorporated by reference). In this case, insulinis extracted from mouse pancreas and its concentration is determined byits immunoreactivity, such as by radioimmunoassay techniques, usingmouse insulin as a standard.

[0240] In addition to monitoring NOD mice for diabetes in general, theeffects of the inventive methods of treatment are also monitored forgene-specific or gene product-specific effects if the stem cellsadministered were transformed or transfected with a heterologous gene,thereby allowing a correlation to be drawn between expression of theheterologous gene and its effects on diabetes. For example, the presenceof the heterologous gene product may be examined by immunohistochemistryof the pancreatic β-cells of NOD mice for the gene product and forinsulin. The expression of the patched and smoothened genes is furtherexamined in NOD mouse islets by detection of the RNA transcript for thepatched and smoothened receptors. Reverse transcription-polymerase chainreaction (RT-PCR) amplification is performed by known means to amplify afragment of mouse patched or smoothened cDNA, and analyzed by agarosegel electrophoresis, according to standard means. The identification ofthe amplified cDNA fragment is confirmed as corresponding to the patchedor smoothened RNA by hybridization of the amplified fragment with aradiolabeled internal oligonucleotide probe for the patched orsmoothened genes, or by other such methods as known to one skilled inthe art.

EXAMPLE 5

[0241] Immunocytochemical Identification of Nestin Positive Human andRat Pancreatic Stem Cells

[0242] Pancreatic islets were analyzed for nestin expression. Islets andstem cells were isolated as described above. Nestin expression wasobserved by immunocytochemical staining in a distinct population ofcells within developing islet clusters of embryonic day 16 (E16) ratpancreas (FIG. 8A) and in islets of the adult pancreas (postnatal 60days) (FIG. 8B). Immunocytochemical staining was performed as follows.

[0243] Cryosections (6 μM) prepared from embryonic day 16 and adult (60day) rat pancreata as well as cells were fixed with 4% paraformaldehydein phosphate. Cells were first blocked with 3% normal donkey serum for30min at room temperature and incubated with primary antisera overnight at4° C. The antisera were rinsed off with PBS and incubated with therespective Cy-3 and Cy-2 labeled secondary antisera. for 1 hour at roomtemperature. Slides were then washed with PBS and coverslipped withfluorescent mounting medium (Kirkegaard and Perry Labs, Gaithersburg,Md.). Tissue sections were incubated overnight at 4° C. with primaryantisera. Primary antisera were then rinsed off with PBS, and slideswere blocked with 3% normal donkey serum for 10 min at room temperaturebefore incubation with donkey anti-Cy3 (indocarbocyanine) and eitheranti-guinea pig (insulin), anti-mouse (glucagon), or anti-sheep(somatostatin) sera DTAF (Jackson Immuno Research Laboratories, WestGrove, Pa.) for 30 min at room temperature. Slides were then rinsed withPBS and coverslipped with fluorescent mounting medium (Kirkegaard andPerry Laboratories, Gaithersburg, Md.). Fluorescence images wereobtained using a Zeiss Epifluorescence microscope equipped with anOptronics TEC-470 CCD camera (Optronics Engineering, Goleta, Calif.)interfaced with a PowerMac 7100 installed with IP Lab Spectrum analysissoftware (Signal Analytics Corp, Vienna, Va.).

[0244] The nestin-positive cells are distinct from β-, α-, δ-, andPP-cells because they do not co-stain with antisera to the hormonesinsulin (FIG. 8A & B), glucagon, somatostatin, or pancreaticpolypeptide. The nestin-positive cells also do not co-stain withantisera to collagen IV, a marker for vascular endothelial cells (FIG.8C) nor with an antiserum to galanin, a marker for nerve cells or amonoclonal antibody to cytokeratin 19, a specific marker for ductalcells (FIG. 8). Nestin-positive staining is associated with distinctcells within the islets clearly observed by nuclear containing (FIG.4D).

EXAMPLE 6

[0245] Identification of Nestin Positive Human and Rat Stem Cells byRT-PCR

[0246] To confirm the immunocytochemical identification of nestinexpression in pancreatic islets, we performed an RT-PCR of the nestinmRNA using total RNA prepared from freshly isolated rat islets and humanislet tissue. RT-PCR was performed according to the following method.

[0247] Total cellular RNA prepared from rat or human islets was reversetranscribed and amplified by PCR for 35 cycles as described previously(Daniel et al., 1998, Endocrinology, 139:3721-3729). Theoligonucleotides used as primers or amplimers for the PCR and as probesfor subsequent Southern blot hybridization are: Rat nestin: Forward,5′gcggggcggtgcgtgactac3′; Reverse, 5′aggcaagggggaagagaaggatgt3′;Hybridization, 5′aagctgaagccgaatttccttgggataccagagga3′. Rat keratin 19:Forward, 5′acagccagtacttcaagacc3′; Reverse, 5′ctgtgtcagcacgcacgtta3′;Hybridization, 5′tggattccacaccaggcattgaccatgcca3′. Rat NCAM: Forward,5′cagcgttggagagtccaaat3′; Reverse, 5′ttaaactcctgtggggttgg3′;Hybridization, 5′aaaccagcagcggatctcagtggtgtggaacgatgat3′. Rat IDX-1Forward, 5′atcactggagcagggaagt3′ Reverse, 5′gctactacgtttcttatct3′Hybridization, 5′gcgtggaaaagccagtggg3′ Human nestin: Forward,5′agaggggaattcctggag3′; Reverse, 5′ctgaggaccaggactctcta3′;Hybridization, 5′tatgaacgggctggagcagtctgaggaaagt3′. Human keratin:Forward, 5′cttttcgcgcgcccagcatt3′; Reverse, 5′gatcttcctgtccctcgagc3′;Hybridization, 5′aaccatgaggaggaaatcagtacgctgagg3′. Human glucagon:Forward, 5′atctggactccaggcgtgcc3′; Reverse, 5′agcaatgaattccttggcag3′;Hybridization, 5′cacgatgaafttgagagacatgctgaaggg3′.

[0248] Primers were selected from two different exons and encompassed atleast one intronic sequence. In addition, an RT minus control was runfor most samples. PCR cycling was at 94° C. for 1 min followed by 94° C.for 10 secs, 58/56° C. for 10 secs, 72° C. for 1 min, 35 cycles, and 72°C. for 2 min. The annealing temperature was 58° C. for rat nestin and56° C. for the remaining primer pairs.

[0249] For Southern hybridization oligonucleotide probes wereradiolabeled with T4 polynucleotide kinase and γ³²P ATP. Radiolabeledprobes were hybridized to PCR products that had been transferred tonylon membranes at 37° C. for one hour, then washed in 1×SSC+0.5% SDS at55° C. for 10-20 min or 0.5×SCC+0.5% SDS at 42° for the human PCRproducts.

[0250] The RT-PCR generated products of the correctly predicted size(FIG. 8E, upper panels) and were confirmed by Southern blotting (FIG.8E, lower panels) and by DNA sequencing of the products. These datademonstrate the identification of a new cell type in pancreatic isletsthat expresses nestin and may represent an islet pluripotential stemcell similar to the nestin-positive stem cells in the central nervoussystem.

EXAMPLE 7

[0251] The ATP-dependent Transporter ABCG2 is Expressed inNestin-positive Cells Derived from Pancreatic Islets

[0252] Human islet-derived NIPs contain a substantial subpopulation ofSP cells that co-express ABCG2, MDR1 and nestin. Nestin was first shownto be a marker of neural and muscle stem/progenitor cells (Lendahl, etal., 1990, Cell 60:585: Zimmerman et al.; 1994, Neuron 12:11). Neuralstem cells especially exhibit a high degree of plasticity giving rise toneurons and different types of glial cells. Neural stem/progenitor cellsalso differentiate into hematopoietic cells, which would make them trulypluripotential stem cells (Shih et al., 2001, Blood 98:2412). The bestexamples of pluripotential adult stem cells to date are those derivedfrom the bone marrow, so called side population (SP) cells (Goodell etal., 1996, J Exp Med 183:1797). SP cells have been identified by theirproperty to effectively exclude the fluorescent vital dye Hoechst 33342and contain the vast majority of bone marrow repopulating cells. SPcells represent 0.05% of the whole bone marrow of adult humans and mice,and apart from giving rise to all hematopoietic lineages they can alsogive rise to skeletal and cardiac muscle as well as to endothelial cells(Gussoni et al., 1999, Nature 401:390; Jackson et al., 2001, J ClinInvest 107:1395). The ATP-binding cassette transporter ABCG2 (BCRP1) hasbeen demonstrated to be a major component of the SP phenotype, thusoffering a molecular means to specifically identify SP-cells (Kim etal., 2002, Clin Cancer Res 8:22; Scharenberg et al., Blood 99:507; Zhouet al., 2001, Nat Med 7:1028). Notably, marrow-derived SP cells have alimited, if any, capacity to proliferate in vitro (Bunting et al., 2000,Blood 96:902; Storms et al., 2000, Blood 96:2125), but enforcedP-glycoprotein pump (MDR-1) function in bone marrow cells results in theexpansion of SP stem cells in vitro and repopulating cells in vivo(Bunting et al., 2002, Stem Cells 20:11).

[0253] NIPs, linked to neural stem cells by their expression of nestin,also show characteristics of bone marrow SP stem cells, by virtue oftheir expression of ABCG2 in a substantial subpopulation of NIPsidentified by the Hoechst 33342 exclusion assay. This side population ofNIPs also expresses MDR-1, contributing to their continued expansion invitro. Thus NIPs may be a potential source of adult pluripotentialstem/progenitor cells useful for the production of islet tissue fortransplantation into diabetic subjects.

[0254] Human pancreatic islets were obtained from the islet distributionprograms of the Cell Transplant Center, Diabetes Research Institute,University of Miami School of Medicine (Miami, Fla.), and the JuvenileDiabetes Research Foundation Center for Islet Transplantation, HarvardMedical School (Boston, Mass.). NIPs were isolated as previouslydescribed (Zulewski et al., 2001, Diabetes 50:521). Briefly, humanpancreatic islets were handpicked, incubated at 37° C. at 5% CO₂ inconcanavalin A (ConA) coated culture dishes in modified RPMI 1640 mediasupplemented with 10% fetal bovine serum, 10 mmol/l HEPES buffer, 1mmol/l sodium pyruvate, 71.5 μmol/l β-mercaptoethanol andantibiotic-antimycotic (Gibco Life Technologies, Gaithersburg, Md.).After 96 hours floating islets were transferred into fresh media furtherthat was supplemented with 20ng/ml basic fibroblast growth factor (bFGF)and 20ng/ml epidermal growth factor (EGF) (both from Sigma, St. Louis,Mo.) in new plastic culture dishes without ConA coating. The isletsattached to the uncoated plastic surface and within several days amonolayer of cells was observed growing out and away from the islets.Cells from the periphery of the monolayer, nestin-positive islet-derivedprogenitor cells (NIPs), were transferred to a new culture dish andgrown to near confluence under the same conditions as above. Cells werefurther split into two culture dishes to perform Hoechst 33342 dyeexclusion assays with and without verapamil, an inhibitor of H33342 dyetransport (Goodell et al., 1996, supra).

[0255] RT-PCR demonstrated the expression of ABCG2 (FIG. 19A). TheRT-PCR-generated DNA product was sequenced and shown to be identical(586 bps) with that of the human ABCG2 (GenBank Accession No. XM-032424,FIG. 18)) (Data not shown). Southern blot hybridization with a clonedABCG2 probe confirmed the amplification of the correct cDNA (FIG. 19A).

[0256] RNA was isolated from cultured NIPs as well as from sorted SPcells and non-SP controls (5000 and 10,000 cells respectively) usingTrizol (Gibco) following the manufacturers protocol. Single strandedcDNA was made with the Superscript First-Strand System (Invitrogen,Carlsbad, Calif.). The cDNAs were amplified by polymerase chain reaction(PCR). Controls without reverse transcriptase (-RT controls) were donefor all PCR reactions. PCR products were analyzed by agarose gelelectrophoresis and the correct identity of products was confirmed bysequencing. Template concentrations were normalized for GAPDH (31cycles). ABCG2, MDR1 and nestin were amplified with 34, 38, and 36cycles respectively. Primers were: 5′tgaaggtcggagtcaacggatttggt3′ and5′catgtgggccatgaggtccaccac3′ for GAPDH, 5′agaggggaattcctggag3′ and5′ctgaggaccaggactctcta3′ for nestin, 5′tcctggagcggttctacgac3′ and5′gggcttcttggacaaccttttca3′ for MDR1 and 5′gctggggttctcttcttcctgacg3′and 5′ctaccccagccagtgtcaac3′ for ABCG2. PCR products for ABCG2 weretransferred to nylon membranes (Hybond N+; Amersham Pharmacia; LittleChalfont; GB). Blots were hybridized with radiolabeled, cloned ABCG2 orMDR1 probes using Rapid Hyb Buffer (Amersham) following themanufacturers protocol. Primers for the full open reading frame of humanABCG2 were 5′-attaagctgaaaagataaaaactctcc3′ and5′atgtgaggataaatcatactgaat3′ (base pairs 174-202 and 2184-2207 ofGenbank sequence XM_(—)032424).

EXAMPLE 8

[0257] 1.5 to 2% of Cultured Cells Have a Side Population Phenotype inthe Hoechst 33342 Dye Exclusion Assay

[0258] NIPs, linked to neural stem cells by their expression of nestin,also show characteristics of bone marrow SP stem cells, by virtue oftheir expression of ABCG2 in a substantial subpopulation of NIPsidentified by the Hoechst 33342 exclusion assay. This side population ofNIPs also expresses MDR-1, contributing to their continued expansion invitro.

[0259] To investigate whether the expression of ABCG2 lends a sidepopulation (SP) phenotype to some of the cultured NIPs, Hoechst 33342dye exclusion assays were performed following the published protocol(Goodell et al., 1996, supra) with slight modifications. The exclusionof the Hoechst 33342 dye, which defines the pluripotential sidepopulation (SP) of hematopoietic stem cells, is mediated by theATP-binding cassette transporter, ABCG2. Verapamil was used as aspecific inhibitor of H33342 transport (Goodell et al., 1996, supra).

[0260] The Hoechst dye exclusion assay was done following the protocolof Goodell et al. 1996, supra, with the modification that staining wasperformed while the cells were still attached to the culture dish. Cellviability with this change in protocol was significantly higher comparedto the original protocol in which cells are stained while in suspension.Hoechst 33342 (Sigma) was added to the growth media at a finalconcentration of 5 μg/ml and cells were incubated for 90 minutes at 37°C. Where indicated, verapamil (Sigma) was added 15 minutes prior to thestart of the assay at a final concentration of 50 μmol/l. For analysiscells were trypsinized, poured through a 40 μm cell strainer, andresuspended in ice cold Hanks' balanced salt solution containingpropidium iodide (Sigma) at a final concentration of 2 μg/ml for thediscrimination of dead cells. Flow cytometry was done on a Vantage and aMoflow cell sorter using a UV laser for excitation of Hoechst 33342 andpropidium iodide. 450/20 BP (blue) and 630/20 (red) filters were usedfor analysis in linear mode.

[0261] Fluorescence activated cell sorting (FACS) showed a clearlyvisible side population (FIG. 19B; 2.1% gated cells) which was absent inthe presence of the inhibitor verapamil (FIG. 19C; 0.1% gated cells). Inanalyses of two other NIP cultures independently derived, the percentageof SP-positive cells was 1.5 and 2 percent, respectively (data notshown). We also amplified the full open reading frame of ABCG2 fromNIPs. Cloning into an expression vector and transfection into INS-1insulinoma cells resulted in effective Hoechst 33342 exclusion intransfected cells (data not shown).

EXAMPLE9

[0262] The SP Phenotype Correlates with the Expression of ABCG2, MDR1,and Nestin

[0263] SP cells and non-SP control cells were isolated from culturedNIPs by FACS (FIG. 20A; gates R1 and R2 respectively). Expression ofABCG2 correlated well with the SP phenotype as shown by RT-PCR (FIG.20B) and Southern blot hybridization with a cloned ABCG2 probe. RT-PCRand Southern blot hybridization demonstrated expression of MDR1((P-glycoprotein), another ATP-binding cassette transporter present inhematopoietic stem cells (Chaudhary et al., 1991, Cell 66:85)) in theSP-fraction of cultured NIPs (FIG. 20B). The SP fraction also expressednestin at a markedly higher level than the non-SP controls (FIG. 20B.

[0264] Nestin-positive cells derived from human pancreatic isletscontain 1.5 to 2% of SP cells, which express ABCG2 and nestin at highlevels compared to non-SP control cells. The correlation of ABCG2expression with the SP phenotype confirms the finding that ABCG2activity is a major component of the SP dye efflux (Kim et al., supra;Scharenberg et al., supra; Zhou et al., supra). The coexpression ofnestin and ABCG2 indicates a broader role for nestin as a general markerof stem/progenitor cells.

[0265] The SP cell fraction of the bone marrow in different mammalianspecies including humans contains the vast majority of multipotentialhematopoietic stem cells (HSCs) (Goodell et al., 1997, supra). Thedifferentiation of bone marrow-derived SP cells is not limited to bloodlineages; in animal models SP cells also differentiate to skeletal andcardiac muscle as well as endothelial cells (Gussoni et al., Nature401:390; Jackson et al., 2001, J Clin Invest 107:1395). In addition,bone marrow-derived stem cells differentiate into functional brain andliver cells (Brazelton et al., 2000, Science 290:1775; Lagasse et al.,2000, Nat Med 6:1229).

[0266] The portion of SP cells in the NIP cultures (1.5 to 2%) is atleast 20-fold higher than that found in the bone marrow (0.05%) (Goodellet al., 1996, supra). However, a comparable percentage of SP cells isfound in cultures of muscle satellite cells, which are considered to bemyogenic progenitors (Jackson et al., 1999, Proc Natl Acad Sci96:14482). In addition to generating differentiated myofibres muscle SPcells have been shown to reconstitute the bone marrow of lethallyirradiated mice, thus exhibiting an unexpected degree of plasticity(Gussoni et al., 1999, supra). Some evidence exists that these SP cellsalso come from the bone marrow before taking up residence in the muscle(Kawada et al., 2001, Blood 98:2008). An SP population has also beendemonstrated in mouse embryonic stem cells (Zhou et al., 2001, supra).The expression on ABCG2 neural stem cells has been shown to besignificantly higher than it is in more differentiated neuralcells(Geschwind et al., 2001, Neuron 29:325).

[0267] MDR1 (P-glycoprotein, ABCB1) is another ATP-binding cassettetransporter expressed in hematopoietic stem cells (among several othercell types) (Zhou et al., 2001, supra; Chaudhary et al., 1991, supra).Overexpression of MDR1 in bone marrow cells leads to an expansion of theSP population, their prolonged survival in culture and an enhancedrepopulating activity after transplantation into mice (Bunting et al.,2000, supra). In some of the transplanted animals it ultimately resultsin a myeloproliferative syndrome resembling chronic myelogenous leukemia(Bunting et al., 1998, Blood 92:2269). Therefore MDR-1 expression hasbeen implicated in the expansion of hematopoietic stem cells and wassuggested as a characteristic of proliferating stem cells in contrast toquiescent cells, only expressing ABCG2 (Bunting et al., 2002, supra).MDR1 is expressed in the SP fraction of pancreatic islet derived NIPs.

EXAMPLE 10

[0268] Immunocytochemical Identification of GLP-1R-positive HumanPancreatic Stem Cells

[0269] Nestin-positive pancreatic islet stem cells were analyzed forGLP-1R expression. Human islet tissue was obtained from the JuvenileDiabetes Research Center for Islet Transplantation, Harvard MedicalSchool. NIPs were isolated as described previously (Zulewski, et al.,2001, Diabetes 50: 521). Briefly, islets were washed and cultured inRPMI 1640 medium containing serum, 11.1 mM glucose, antibiotics, sodiumpyruvate, β-mercaptoethanol, and growth factors. Within several days,nestin-positive cells (identified immunocytochemically as describedabove) grew out from the islets. Later, these cells were cloned andexpanded in medium containing 20 ng/ml of basic fibroblast growth factorand epidermal growth factor or 1000 units of recombinant human leukemiainhibitory factor. Incubation with GLP-1 was administered in the absenceof serum and fresh peptide was added every 48 h without changing themedium.

[0270] Immunocytochemical detection of GLP-1R was performed using rabbitpolyclonal antisera (Heller et al., supra) as follows. Cells cultured onLab-Tek chamber slides (Nunc, Naperville, Ill.) or gridded coverslips(Bellco Glass, N.J.) were fixed with 4% paraformaldehyde in PBS for 10minutes at room temperature. After several rinses in PBS, cells wereblocked with normal donkey serum for 30 minutes and incubated withprimary antisera or pre-immune sera at 4° C. The following day, cellswere rinsed with PBS and incubated with secondary antisera (donkeyanti-rabbit and donkey anti-guinea pig) labeled with Cy-3 or Cy-2 for 1hour at room temperature. After several washes, coverslips containingcells were mounted onto slides in mounting medium containing DAPI(Vector Laboratories, Burlingame, Calif.) which stains nuclei.Fluorescence images were obtained using a Zeiss epifluorescencemicroscope equipped with an Optronics TEC-470 CCD camera (OptronicsEngineering, Goleta, Calif.) interfaced with a Powermac 7100. IP labSpectrum software (Signal Analytics, Vienna, Va.) was used to acquireand analyze images.

[0271] GLP-1R immunoreactivity was detected in the majority of NIPs (atleast 60%) examined (FIG. 22A).

EXAMPLE 11

[0272] Identification of GLP-R1-positive Human Stem Cells by RT-PCR

[0273] To confirm the immunocytochemical identification of GLP-1Rexpression in pancreatic islets (Nips), RT-PCR was performed using oftotal RNA prepared from NIPs. RT-PCR was performed as described above inexample 6, for the identification of nestin mRNA with the differencebeing that the following GLP-1R-specific primers were used: 5′gtgtggcggccaattactac 3′ (Forward); 5′ cttggcaagtctgcatttga 3′ (Reverse).Amplification of NIP mRNA produced the expected 346 bp product (FIG.22B) indicating that, in addition to expressing the GLP-1R protein, NIPshave the biosynthetic ability to produce GLP-1R. GLP-1R, therefore, inaddition to nestin, is useful in the present invention as a marker forpancreatic stem cells.

EXAMPLE 12

[0274] In vitro Proliferation of Nestin Positive Stem Cells

[0275] The ability of nestin-positive stem cells to proliferate in vitrowas determined.

[0276] Islets prepared from 60 day-old rats or a normal adult human werefirst plated on concanavalin-A-coated dishes and cultured in modifiedRPMI 1640 medium containing 10% fetal bovine serum for four days topurge the islet preparation of fibroblasts and other non-islet cellsthat adhered to the ConA-coated plates. The islets that did not adhereto the plates under these culture conditions were collected andtransferred to 12-well plates (without ConA coating) containing the samemodified RPMI 1640 medium now additionally supplemented with bFGF andEGF (20 ng/mL each). The growth factors bFGF and EGF together wereselected because they are known to stimulate the proliferation of neuralstem cells derived from ependyma of the brain (Reynolds and Weiss, 1996,Dev. Biol., 175:1-13). The islets attached to the plates and cellsslowly grew out of the islet as a monolayer (estimated cell doublingtime 40-45 hrs in human cells). The outgrowing monolayer of cells werephenotypically homogenous (FIG. 9A, panel 1) and expressed nestin (FIG.9A, panel 2). Rat cells were picked from the monolayer (batches of atleast 20-30 cells), subcloned into 12-well plates, and incubated withthe modified RPMI 1640 medium (11.1 mM glucose) containing bFGF and EGF.The subcloned cells grew rapidly and became confluent at six days withan estimated cell doubling time of 12-15 hrs (FIG. 9A, panel 3), and by12 days formed wave-like structures. After 15-17 days of culture, thecells formed islet-like clusters (ILCs) (FIG. 9A, panel 4). Similarcells were cloned from human islets (FIG. 9B). Upon reaching confluence(FIG. 9B, panel 1), the human cells migrated to form large vacuolatedstructures in the dish (FIG. 9B, panels 2 and 3). The cells lining thelarge spaces then changed morphology, rounded, and aggregated togetherforming three dimensional ILCs (FIG. 9B, panels 4-6).

[0277] Indicators of differentiation of these nestin-positive isletprogenitor cells (NIPs) that formed these ILCs were characterized byRT-PCR and Southern blot and found that they express the endocrinemarker NCAM (neural cell adhesion molecule) (Cirulli et al., 1994, J.Cell Sci., 107:1429-36) (FIG. 9C, right panel) and the ductal cellmarker CK19 (cytokeratin 19) (Bouwens et al., 1998, J. Pathol.,184:234-9; Bouwens et al., 1995, J. Histochem. Cytochem., 43:245-53;Bouwens et al., 1994, Diabetes, 43:1279-93) (FIG. 9C, left panels). Atthis stage of the studies it was concluded that when the NIPs becameconfluent and aggregated into islet-like cell clusters, they began toexpress pancreatic genes (NCAM and CK19), but were limited in expressionof islet genes because of the absence of growth factors essential fortheir differentiation to endocrine cells. It was also recognized thatthe differentiation of a progenitor cell population typically requiresfirst a proliferative phase and then quiescence of proliferation in thepresence of differentiation-specific morphogen growth factors. Thereforethe culture conditions were modified in some instances by replacing themedia containing 11.1 mM glucose, bFGF and EGF, which inducesproliferation of cells, with media containing lower glucose (2.5 mM),which is less proliferative, and the factors HGF/Scatter Factor orbetacellulin and Activin A. Glucose is a known proliferative factor forpancreatic islet β-cells (Swenne, 1992, Diabetologia, 35:193-201;Bonner-Weir, 1989, Diabetes, 38:49-53) and both HGF/Scatter Factor andActivin A have been shown to differentiate the pancreatic ductal cellline AR42J into an endocrine phenotype that produces insulin, glucagon,and other pancreatic endocrine cell proteins (Mashima et al., 1996,Endocrinology, 137:3969-76; Mashima et al., 1996, J. Clin. Invest.,97:1647-54).

[0278] Cultures containing ILCs expressed the pancreas-specifichomeodomain protein IDX-1 by immunocytochemistry (FIG. 10A, upperpanel), RT-PCR and Southern blot (FIG. 10B), and by Western immunoblot(FIG. 10C). The ILCs also expressed the mRNA encoding proglucagon asseen by RT-PCR (FIG. 10D) and produced immunoreactive glucagon,glucagon-like peptide-1, and insulin. Radioimmunoassays of mediaobtained following 72-96 h of culture of islet-like clusters in severalwells gave values of 40-80 pg/ml GLP-1, 30-70 pg/ml glucagon, 29-44pg/ml insulin. Radioimmunoassays were performed as follows.

[0279] Insulin and glucagon concentrations in culture media weredetermined by ultra sensitive radioimmunoassay kits purchased from LincoResearch Inc. and DPC Inc., respectively. The antisera supplied in therespective kits are guinea pig anti-human insulin and rabbit anti-humanglucagon. GLP-1 secretion was measured with an anti-humanGLP-1(7-36)amide rabbit polyclonal antiserum raised by immunization of arabbit with a synthetic peptide CFIAWLVKGR amide conjugated to keyholelimpet hemocyanin. The antiserum is highly specific for the detection ofGLP-1(7-36)amide and only weakly detects proglucagon. The sensitivitylevels for these assays are 6 pg/mL, 13 pg/mL and 10.2 pg/niL,respectively.

[0280] Incubation of the ILCs for 7 days in 10 mM nicotinamide, asdescribed by Ramiya et al. (Ramiya et al., 2000, Nat. Med., 6:278-282),increased insulin secretion by 2- to 3-fold.

[0281] Several additional pancreatic markers were expressed indifferentiated NIPs such as glucose transporter-2 (Wang et al., 1998),synaptophysin, and HGF (Menke et al., 1999) as shown in FIG. 15. Todetermine whether the differentiating NIPs may have properties ofpancreatic exocrine tissue, we used RT-PCR and detected the expressionof amylase and procarboxypeptidase (FIG. 15).

[0282] Some cultures of NIPs containing stem cells also expressed themRNA encoding proglucagon and insulin as seen by RT-PCR (FIG. 16A andB).

[0283] The expression of IDX-1 is of particular importance because it isrecognized to be a master regulator of pancreas development, andparticularly to be required for the maturation and functions of thepancreatic islet β-cells that produce insulin (Stoffers et al., 1997,Trends Endocrinol. Metab., 8:145-151).

[0284] Because the neogenesis of new islets is also known to occur bydifferentiation of cells in pancreatic ducts, particularly during theneonatal period (rats and mice) but to some extent throughout adult life(Bonner-weir et al., 1993, Diabetes, 42:1715-1720; Rosenberg, 1995, CellTransplant, 4:371-383; Bouwens et al., 1996, Virchows Arch.,427:553-560), nestin expression was analyzed in the pancreatic ducts ofadult rats. By dual fluorescence immunocytochemistry with antisera tonestin and to cytokeratin 19, a marker of ductal epithelium, nestin isstrongly expressed in localized regions of both the large and smallducts, as well as in some centrolobular ducts within the exocrine acinartissue (FIGS. 11A and 11B). Remarkably, the localized regions of nestinexpression in the ducts are mostly devoid of staining with the anti CK19antiserum. Further, the nestin-positive cells in the ducts appear tohave a morphology that is distinct from that of the epithelial cells.The epithelial cells consist of a homogenous population of cuboidal,rounded cells, whereas the nestin-positive cells are nucleated,serpiginous and appear to reside in the interstices among or aroundepithelial cells (FIG. 11C).

[0285] Thus, CK19 is not expressed in the majority of ductal cells thatexpress nestin suggesting that these nestin-expressing cells locatedwithin the pancreatic ducts are a passenger population of cells distinctfrom the ductal epithelial cells and are stem cells that have not yetdifferentiated into a ductal or endocrine phenotype. The finding oflocalized populations of nestin-expressing cells within the pancreaticducts and islets of the adult rat pancreas further supports the ideathat rat pancreatic ducts contain cells that are progenitors of isletcells (neogenesis), but these progenitors are not a subpopulation ofductal epithelial cells per se.

EXAMPLE 13

[0286] Transplantation of Pancreatic Stem Cells Engineered to ExpressIDX-1 in Human Subjects with Diabetes Mellitus

[0287] Islets isolated from pig or human donor pancreata, or frompancreatic biopsy of eventual human transplant recipient are cultured exvivo in conditions that stimulate outgrowth of stem cells. Stem cellsare then isolated away from islets (cloned), expanded in vitro inproliferation media containing bFGF-2, EGF, and 11.1 mM glucose,transfected/injected with an expression vector containing DNA encodingtranscription factor IDX-1, and transplanted into a diabetic recipient.Alternatively, IDX-1-transfected stem cells are treated with GLP-1, orother differentiation morphogens or growth factors for 1-3 days beforetransplantation to initiate processes of differentiation of engineeredstem cells to β-cells. In one embodiment, stem cells are neitherexpanded or differentiated prior to administration to the recipient orare only expanded or differentiated prior to administration to therecipient. In one embodiment, GLP-1 is administered to the recipientduring and for several days after transplantation to stimulatedifferentiation of stem cells and encourage successful engraftment.According to this method, xenographs (pig islets) or allographs (humanislets from a human donor that is not the recipient), as well asisographs (islets derived from the recipient) are carried out. It ishypothesized that when transplanted to a host recipient the stem cellgenetic repertoire is reprogrammed so that the host recognizes the stemcells (in the case of xenographs or allographs) as self, such thatimmune intolerance and graft rejection and destruction by autoimmunity(type 1 diabetes) does not occur.

EXAMPLE 14

[0288] Transplantation of Pancreatic Stem Cells Cultured to StimulateExpression of IDX-1 in Human Subjects with Diabetes Mellitus

[0289] Islets isolated as described are cultured ex vivo for severaldays in conditions that stimulate first the expansion (proliferation) ofstem cells that exist within the islets and then the expression oftranscription factor IDX-1. The proliferation of stem cells is achievedby culturing the islets in media containing bFGF-2, EGF, and 11.1 mMglucose. Induction of the expression of IDX-1 is achieved by incubationin the presence of GLP-1 and 2 mM glucose. The islets so preconditionedby the treatments described are transplanted to the host recipient.Additionally, the host recipient may be administered GLP-1 during andfor several days after the transplantation to further expand anddifferentiate stem cells to insulin-producing cells to enhance successof engraftment.

[0290] According to this method, xenographs (pig islets), allographs(human islets from a human donor that is not the recipient), as well asisographs (islets derived from the recipient) are effectuated.

EXAMPLE 15

[0291] Xenogeneic Transplantation of Pancreatic Stem Cells into theKidney

[0292] Human nestin-positive-islet progenitor cells (NIPS) were isolatedas described, and transplanted under the renal capsules of eight C57B16mice that were not immunosuppressed. The transplanted human cells werenot rejected by the mouse recipient. Current understanding is that axenograft, such as human tissue, would be rejected by the mouse within5-10 days. Contrary to current understanding, we found that in 8 of the8 non-immunosuppressed mice tested to date, all of the transplantssuccessfully engrafted and proliferated into large masses of tissueengulfing the pole of the kidney by one month (30-38 days) after atransplantation of approximately 10⁵ to 10⁶ cells.

[0293] One C57B16 mouse was sacrificed and determined to have a largearea of new growth at the site of transplantation. A section of thekidney that included the new tissue was divided into two pieces; onepiece was frozen for frozen section histology, and the other piece wasfixed in paraformaldehyde for paraffin section histology. Frozensections were prepared and stained with hematoxylin and eosin (H&E) andantisera to various islet cell antigens.

[0294] Examination of the H&E stained kidney section demonstrated thepresence of a new growth that was not part of the kidney, exhibiting apleiomorphic morphology consisting of a mixed mesenchymal and epithelialtissue containing hepatic, neural, ductal, adipodipic and hematopoeticcomponents. Photomicrographs of the kidney section demonstrated that thenew growth seemed to be invading the renal parenchyme, and theglomeruli. Specific immunostaining with human-specific (not mouse)antisera revealed cords of immunopositive cells staining forhuman-specific keratins, vimentin, and the CD45 leukocyte antigenspecific for human hematapoetic lymphocytes. The kidney of a secondC57B16 mouse also had a similar looking new growth at the site of theNIP transplantation.

[0295] The paraffin section of the NIP-engrafted kidney of a C57B16mouse (the first mouse to be sacrificed) was examined. The tissue blockthat was examined was from the top of the kidney and showed the foreigntissue to be well contained under the renal capsule with no signs of“invasion” into the renal parenchyma. Notably, amongst thepleiomorphic-looking graft tissues were areas that resembled renalparenchyma. Without being bound to theory one hypothesis is that thegraft consists of stem cells trying to differentiate and that the stemcells are not “invading” but simply migrating and proliferating andlooking for a niche, i.e. mesenchymal instructions. They may bereceiving cues from the kidney and may be attempting to differentiateinto kidney. The graft cells may not be malignant, but may be just stemcells attempting to carry out their function.

EXAMPLE 16

[0296] Xenogeneic Transplantation of Pancreatic Stem Cells into thePancreas

[0297] Human nestin-positive-islet progenitor cells (NIPS) are isolatedas described, and transplanted into the pancreas of mice that are notimmunosuppressed and are (a) injured by streptozotocin (to producestreptozotocin induced diabetes) treatment or (b) NOD mice in whichthere is an ongoing islet is with inflammation.

[0298] The pancreas of the transplanted animals is examined to determineif the NIPs find their proper niche, receive instructions from the isletregion, and differentiate into islet (β-cell) cells.

EXAMPLE 17

[0299] Treatment of Diabetes by Xenogeneic Transplantation of PancreaticStem Cells

[0300] Human islets are isolated as described and cultured for severaldays in vitro to expand the stem cell population. Human NIPS aretransplanted to the liver via the portal vein (according to conventionalprocedures well known in the art for transplantation to the liver.

[0301] Alternatively, a population of human NIPs (isolated as described)are introduced into the blood stream. In certain embodiments, the humanNIPS are introduced via the pancreatic artery, to direct them to thediabetic pancreas.

[0302] A population of control (untransplanted animals) and transplantedanimals are analyzed for amelioration of the symptoms of diabetes (e.g.blood glucose levels, insulin levels, number of pancreatic β-cells.

EXAMPLE 18

[0303] Identification of Nestin Positive Stem Cells in the Liver

[0304] Rat livers were isolated and frozen section were preparedaccording to methods known in the art and described herein.

[0305] Frozen sections of rat liver (6 μM) were immunostained with arabbit polyclonal anti-nestin serum. The immunofluorescent signal wasdeveloped by reaction of anti-donkey IgG serum tagged with thefluorophore, Cy3 (yellow-orange color. Nestin-positive cells surroundinga possible large biliary duct are depicted in FIG. 13A. Clusters ofnestin positive cells surrounding several small biliary ducts aredepicted in FIG. 13B.

EXAMPLE 19

[0306] Differentiation of NIPs Toward Hepatic Phenotype

[0307] Because of the reported apparent commonalties between hepaticstem cells (oval cells), hepatic stellate cells, and progenitor cells inthe pancreas, and the observations that following some injuries, theregenerating pancreas undergoes liver metaplasia (Slack, 1995; Reddy etal., 1991; Bisgaard et al., 1991; Rao et al., 1996), we performed RT-PCRto detect liver-expressed genes in the stem cells. PCR products wereobtained for XBP-1, a transcription factor required for hepatocytedevelopment (Reimold et al., 2000), and transthyretin, a liver acutephase protein. Several other liver markers were also expressed such asα-fetoprotein (Dabeva et al., 2000), E-Cadherin (Stamatoglou et al.,1992), c-MET (Ikeda et al., 1998), HGF (Skrtic et al., 1999) andsynaptophysin (Wang et al., 1998); see FIG. 15)) The expression ofproteins shared by the pancreas and liver, such as HGF andsynaptophysin, may reflect their common origin from the embryonicforegut endoderm, and represent differentiation toward either pancreaticor hepatic phenotypes.

EXAMPLE 20

[0308] GLP-1R Signalling in NIPs

[0309] The application of GLP-1 amide to single, isolated NIPs elevateslevels of intracellular Ca²⁺ concentration ([Ca²⁺]_(i)). Cells wereplated onto gridded coverslips to permit subsequent immunohistochemicalstaining of the same cells to test for nestin expression. All cells fromwhich [Ca²⁺]_(i) recordings were made were shown to express nestin. Incontrast to adult β-cells, GLP-1 stimulates [Ca²⁺]_(i) levels at basal(5.6 mM) glucose but was ineffective in the presence of high (20 mM)glucose (FIG. 23, panel A). These effects were reproduced by forskolin(FIG. 23, panel B) suggesting that the effects of GLP-1 are mediatedthrough the activation of Gs and cAMP production, the same signallingpathway used in normal β-cells. The pretreatment of single isolated NIPswith the peptide Extendin (9-39), a specific antagonist of GLP-1,prevents the increase in [Ca²⁺]_(i) mediated by GLP-1 (FIG. 23, panels Cand D). The effects of GLP-1R antagonist Extendin (9-39) suggests thatthe same isoform of GLP-1R is expressed in NIPs as in β-cells. The[Ca²⁺]_(i) increase mediated by GLP-1 was inhibited by exprecellularLa³⁺ (5 μM) suggesting that GLP-1 is activating [Ca²⁺]_(i) influx,consistent with its known role to depolarize β-cells (FIG. 23, panel E).We further demonstrate that tolbutamide (100 μM) stimulates [Ca²⁺]_(i)elevation in NIPs indicating that they express ATP-sensitive K+ channels(FIG. 23, panel F). These data are consistent with GLP-1 inducedmembrane depolarization and activation of voltage-dependent Ca²⁺channels in NIPs, consistent with its mechanism of action in β-cells.

EXAMPLE 21

[0310] GLP-1 Induces Differentiation of NIPs into Insulin-secretingCells

[0311] Previous studies have demonstrated the insulinotropic action ofGLP-1 as well as its ability to stimulate β-cell neogenesis in partialpancreactectomized rats (Xu et al., 1999, Diabetes 48:2270). NIPs werepicked from islet cultures and expanded in growth medium containing bFGFplus EGF (passage 01) for 3-10 days (Zulewski et al., 2001 Diabetes 50:521). In certain instances NIPOs that were expanded for 3-5 days werefixed and subjected to immunocytochemical detection of Nestin (Cy-3) andInsulin (Cy-2) as shown in FIG. 24A. It was found that at this stagethey are nestin positive and insulin negative. When NIP cultures wereexpanded for 7-10 days and then treated with either GLP-1 or its stableanalog extendin-4, a subset of cells became insulin positive (Cy-2; FIG.24B, panel 3, 5, and 6) and Idx-1 positive (Cy-3; FIG. 24B, panel 7) andnestin negative (Cy-3; FIG. 23B, panel 4). There was an overall decreasein nestin expression when cultures were treated with GLP-1 (FIG. 24B,panel 2 vs. 4). Accordingly, treatment with Extendin-4 induced a 2- to3-fold increase in insulin secretion (FIG. 25). However, in some culturewells, confluence alone was sufficient to initiate small amounts ofinsulin secretion.

[0312] The homeodomain protein Idx-1 is critical for pancreasdevelopment and plays a major role in the transcriptional regulation ofthe insulin gene. Haploindufficiency of Idx-1 expression results in aform of early onset type-2 diabetes (MODY4) and inherited amino acidchanges in Idx-1 are associated with late onset type-2 diabetes. Idx-1is expressed in differentiated NIP cell populations.

[0313] Human NIPs that had been repeatedly passaged lose their abilityto secrete insuling in response to GLP-1. However, transfection of thesecells with an expression vector encoding the human Idx-1 cDNA renderedthem responsive to GLP-1 and induced the synthesis of insulin in asubset of NIPs (FIG. 26, panel 1). Interestingly, transfection of NIPs(passage 9) with Idx-1 in the absence of GLP-1 treatment did not induceinsulin biosynthesis (FIG. 26, lower panel B), but did stimulate Idx-1expression levels (FIG. 26, upper panel B). Taken together, theseresults suggest that GLP-1 stimulates levels of Idx-1 expression in NIPcells and that a critical threshold concentration of Idx-1 is requiredfor NIPs to convert into insulin-producing cells, thus suggesting a rolefor the GLP-1R in islet cell differentiation.

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OTHER EMBODIMENTS

[0339] Other Embodiments are within the claims that follow.

We claim:
 1. A method of treating a patient with diabetes mellitus,comprising the steps of: (a) isolating a nestin-positive pancreatic stemcell from a pancreatic islet of a donor; and (b) transferring the stemcell into the patient, wherein the stem cell differentiates into aninsulin-producing cell.
 2. The method of claim 1, wherein saidnestin-positive pancreatic stem cell is also ABCG2-positive.
 3. Themethod of claim 1, wherein said nestin-positive pancreatic stem cell isalso positive for at least one of the markers selected from the groupconsisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1,Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2. 4.The method of claim 1, wherein said nestin-positive pancreatic stem celldoes not express at least one of the markers selected from the groupconsisting of CD34, CD45, CD133, MHC class I and MHC class II.
 5. Amethod of treating a patient with diabetes mellitus, comprising thesteps of: (a) isolating an ABCG2-positive pancreatic stem cell from apancreatic islet of a donor; and (b) transferring the stem cell into thepatient, wherein the stem cell differentiates into an insulin-producingcell.
 6. The method of claim 1 or 5, wherein the patient serves as thedonor for said stem cells of step a.
 7. The method of claim 1 or 5,wherein, prior to the step of transferring, the stem cell is treated exvivo with an agent selected from the group consisting of EGF, bFGF-2,high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acidmolecule encoding IDX-1, betacellulin, activin A, TGF-β, andcombinations thereof.
 8. The method of claim 1 or 5, wherein the step oftransferring is performed via endoscopic retrograde injection.
 9. Themethod of claim 1 or 5, additionally comprising the step of: (c)treating the patient with an immunosuppressive agent.
 10. The method ofclaim 9, wherein the immunosuppressive agent is selected from the groupconsisting of FK-506, cyclosporin, and GAD65 antibodies.
 11. A method oftreating a patient with diabetes mellitus, comprising the steps of: (a)isolating a nestin-positive pancreatic stem cell from a pancreatic isletof a donor; (b) expanding the stem cell ex vivo to produce a progenitorcell; and (c) transferring the progenitor cell into the patient, whereinthe progenitor cell differentiates into an insulin-producing beta cell.12. The method of claim 11, wherein said nestin-positive pancreatic stemcell is also ABCG2-positive.
 13. The method of claim 11, wherein saidnestin-positive pancreatic stem cell is also positive for at least oneof the markers selected from the group consisting of Oct3/4, GLP-1receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 andβ1, C-kit, MDR-1, SUR-1, or Kir 6.2.
 14. The method of claim 11, whereinsaid nestin-positive pancreatic stem cell does not express at least oneof the markers selected from the group consisting of CD34, CD45, CD133,MHC class I and MHC class II.
 15. A method of treating a patient withdiabetes mellitus, comprising the steps of: (a) isolating anABCG2-positive pancreatic stem cell from a pancreatic islet of a donor;(b) expanding the stem cell ex vivo to produce a progenitor cell; and(c) transferring the progenitor cell into the patient, wherein theprogenitor cell differentiates into an insulin-producing beta cell. 16.The method of claim 11 or 15, wherein the patient serves as the donorfor said stem cells of step a.
 17. The method of claim 11 or 15, whereinthe step of expanding is performed in the presence of an agent selectedfrom the group consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF,GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1,betacellulin, activin A, TGF-β, and combinations thereof.
 18. The methodof claim 11 or 15, wherein the step of transferring is performed viaendoscopic retrograde injection.
 19. The method of claim 11 or 15additionally comprising the step of: (d) treating the patient with animmunosuppressive agent.
 20. The method of claim 19, wherein theimmunosuppressive agent is selected from the group consisting of FK-506,cyclosporin, and GAD65 antibodies.
 21. A method of treating a patientwith diabetes mellitus, comprising the steps of: (a) isolating anestin-positive pancreatic stem cell from a pancreatic islet of a donor;(b) expanding the stem cell to produce a progenitor cell; (c)differentiating the progenitor cell in culture to form pseudo-islet likeaggregates; and (d) transferring the pseudo-islet like aggregates intothe patient.
 22. The method of claim 21, wherein said nestin-positivepancreatic stem cell is also ABCG2-positive.
 23. The method of claim 21,wherein said nestin-positive pancreatic stem cell is also positive forat least one of the markers selected from the group consisting ofOct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrinsubunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.
 24. The method ofclaim 21, wherein said nestin-positive pancreatic stem cell does notexpress at least one of the markers selected from the group consistingof CD34, CD45, CD133, MHC class I and MHC class II.
 25. A method oftreating a patient with diabetes mellitus, comprising the steps of: (a)isolating an ABCG2-positive pancreatic stem cell from a pancreatic isletof a donor; (b) expanding the stem cell to produce a progenitor cell;(c) differentiating the progenitor cell in culture to form pseudo-isletlike aggregates; and (d) transferring the pseudo-islet like aggregatesinto the patient.
 26. The method of claim 21 or 25, wherein the patientserves as the donor for said stem cells of step a.
 27. The method ofclaim 21 or 25, wherein the step of expanding is performed in thepresence of an agent selected from the group consisting of EGF, bFGF-2,high glucose, KGF, HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acidmolecule encoding IDX-1, betacellulin, activin A, TGF-β, andcombinations thereof.
 28. The method of claim 21 or 25, wherein the stepof transferring is performed via endoscopic retrograde injection. 29.The method of claim 21 or 25 additionally comprising the step of: (e)treating the patient with an immunosuppressive agent.
 30. The method ofclaim 29, wherein the immunosuppressive agent is selected from the groupconsisting of FK-506, cyclosporin, and GAD65 antibodies.
 31. A method ofisolating a stem cell from a pancreatic islet of Langerhans, comprisingthe steps of: (a) removing a pancreatic islet from a donor; (b)culturing cells from the pancreatic islet; and (c) selecting anestin-positive clone from the culture.
 32. The method of claim 31,wherein said nestin-positive clone is also an ABCG2-positive clone. 33.The method of claim 31, wherein said nestin-positive clone is alsopositive for at least one of the markers selected from the groupconsisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1,Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.34. The method of claim 31, wherein said nestin-positive clone does notexpress at least one of the markers selected from the group consistingof CD34, CD45, CD133, MHC class I and MHC class II.
 35. A method ofisolating a stem cell from a pancreatic islet of Langerhans, comprisingthe steps of: (a) removing a pancreatic islet from a donor; (b)culturing cells from the pancreatic islet; and (c) selecting anABCG2-positive clone from the culture.
 36. The method of claim 31 or 35,wherein the culturing is first performed in a vessel coated withconcanavalin A and then again performed in a vessel not coated withconcanavalin A.
 37. The method of claim 31 or 35, comprising theadditional step of: (d) expanding the nestin-positive clone by treatmentwith an agent selected from the group consisting of EGF, bFGF-2, highglucose, KGF, HGF/SF, GLP-1, exendin-4, IDX1, a nucleic acid moleculeencoding IDX-1, betacellulin, activin A, TGF-β, and combinationsthereof.
 38. The method of claim 35 comprising the additional step of:(d) expanding the ABCG2-positive clone by treatment with an agentselected from the group consisting of EGF, bFGF-2, high glucose, KGF,HGF/SF, GLP-1, exendin-4, IDX-1, a nucleic acid molecule encoding IDX-1,betacellulin, activin A, TGF-β, and combinations thereof.
 39. A methodof inducing the differentiation of a nestin-positive pancreatic stemcell into a pancreatic progenitor cell, comprising the step of: treatinga nestin-positive pancreatic stem cell with an agent selected from thegroup consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, anucleic acid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin,activin A, TGF-β, and combinations thereof, whereby the stem cellsubsequently differentiates into a pancreatic progenitor cell.
 40. Themethod of claim 39, wherein said nestin-positive pancreatic stem cell isalso an ABCG2-positive clone.
 41. The method of claim 39, wherein saidnestin-positive pancreatic stem cell is also positive for at least oneof the markers selected from the group consisting of Oct3/4, GLP-1receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 andβ1, C-kit, MDR-1, SUR-1, or Kir 6.2.
 42. The method of claim 39, whereinsaid nestin-positive pancreatic stem cell does not express at least oneof the markers selected from the group consisting of CD34, CD45, CD133,MHC class I and MHC class II.
 43. A method of inducing thedifferentiation of an ABCG2-positive pancreatic stem cell into apancreatic progenitor cell, comprising the step of: treating anABCG2-positive pancreatic stem cell with an agent selected from thegroup consisting of EGF, bFGF-2, high glucose, KGF, HGF/SF, IDX-1, anucleic acid molecule encoding IDX-1, GLP-1, exendin-4, betacellulin,activin A, TGF-β, and combinations thereof, whereby the stem cellsubsequently differentiates into a pancreatic progenitor cell.
 44. Themethod of claim 39 or 43, wherein the pancreatic progenitor cellsubsequently forms pseudo-islet like aggregates.
 45. An isolated,nestin-positive human pancreatic or liver stem cell that is not a neuralstem cell.
 46. The isolated stem cell of claim 45, wherein said cell isalso ABCG2-positive.
 47. The isolated stem cell of claim 45, whereinsaid cell is also positive for at least one of the markers selected fromthe group consisting of Oct3/4, GLP-1 receptor, latrophilin (type 2),Hes-1, Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir6.2.
 48. The isolated stem cell of claim 45, wherein said cell does notexpress at least one of the markers selected from the group consistingof CD34, CD45, CD133, MHC class I and MHC class II.
 49. An isolated,ABCG2-positive human pancreatic or liver stem cell that is not a neuralstem cell.
 50. The isolated cell of claim 49, wherein said cell is alsopositive for at least one of the markers selected from the groupconsisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1,Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.51. The isolated cell of claim 49, wherein said cell does not express atleast one of the markers selected from the group consisting of CD34,CD45, CD133, MHC class I and MHC class II.
 52. The isolated stem cell ofclaim 49, wherein said cell does not express at least one of the markersselected from the group consisting of CD34, CD45, CD133, MHC class I andMHC class II.
 53. The isolated stem cell of claim 45 or 49, thatdifferentiates to form insulin-producing beta cells.
 54. The isolatedstem cell of claim 45 or 49, that differentiates to formglucagon-producing alpha cells.
 55. The isolated stem cell of claim 45or 49, that differentiates to form pseudo-islet like aggregates.
 56. Theisolated stem cell of claim 45 or 49, that differentiates to formhepatocytes.
 57. A method of identifying a pancreatic cell as a stemcell, comprising the step of: contacting a cell with a labelednestin-specific antibody, whereby if the cell becomes labeled with theantibody the cell is identified as a stem cell.
 58. The method of claim57, further comprising the step of contacting a cell with a labeledantibody that binds to a marker selected from the group consisting ofABCG2, Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin,Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2, whereby ifthe cell becomes labeled with the antibody the cell is identified as astem cell.
 59. The method of claim 30 further comprising the step of:contacting the cell with a labeled cytokeratin-19 specific antibody,whereby if the cell does not become labeled with the antibody the cellis identified as a stem cell.
 60. The method of claim 57 furthercomprising the step of: contacting the cell with a labeled collagen IVspecific antibody, whereby if the cell does not become labeled with theantibody the cell is identified as a stem cell.
 61. The method of claim57, further comprising the step of contacting the cell with a labeledantibody that binds to a marker selected from the group consisting of ofCD34, CD45, CD133, MHC class I and MHC class II, whereby if the celldoes not become labeled with the antibody the cell is identified as astem cell.
 62. A method of inducing a nestin-positive pancreatic stemcell to differentiate into hepatocytes, comprising the step of: treatingthe nestin-positive pancreatic stem cell with an effective amount of anagent that induces the stem cell to differentiate into hepatocytes orinto progenitor cells that differentiate into hepatocytes.
 63. Themethod of claim 62, wherein said nestin-positive pancreatic stem cell isalso ABCG2-positive.
 64. The method of claim 62, wherein saidnestin-positive pancreatic stem cell is also positive for at least oneof the markers selected from the group consisting of Oct3/4, GLP-1receptor, latrophilin (type 2), Hes-1, Nestin, Integrin subunits α6 andβ1, C-kit, MDR-1, SUR-1, or Kir 6.2.
 65. The method of claim 62, whereinsaid nestin positive pancreatic stem cell does not express at least oneof the markers selected from the group consisting of CD34, CD45, CD133,MHC class I and MHC class II.
 66. A method of inducing an ABCG2-positivepancreatic stem cell to differentiate into hepatocytes, comprising thestep of: treating the ABCG2-positive pancreatic stem cell with aneffective amount of an agent that induces the stem cell to differentiateinto hepatocytes or into progenitor cells that differentiate intohepatocytes.
 67. The method of claim 62 or 66, wherein the agent iscyclopamine.
 68. A method of treating a patient with liver disease,comprising the steps of: (a) isolating a nestin-positive pancreatic stemcell from a pancreatic islet of a donor; and (b) transferring the stemcell into the patient, wherein the stem cell differentiates into ahepatocyte.
 69. The method of claim 68, wherein said nestin-positivepancreatic stem cell is also ABCG2-positive.
 70. The method of claim 68,wherein said nestin-positive pancreatic stem cell is also positive forat least one of the markers selected from the group consisting ofOct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrinsubunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.
 71. The method ofclaim 68, wherein said nestin positive pancreatic stem cell does notexpress at least one of the markers selected from the group consistingof CD34, CD45, CD133, MHC class I and MHC class II.
 72. A method oftreating a patient with liver disease, comprising the steps of: (a)isolating an ABCG2-positive pancreatic stem cell from a pancreatic isletof a donor; and (b) transferring the stem cell into the patient, whereinthe stem cell differentiates into a hepatocyte.
 73. The method of claim68 or 72, wherein the patient serves as the donor for said stem cells ofstep a.
 74. A method of treating a patient with liver disease,comprising the steps of: (a) isolating a nestin-positive pancreatic stemcell from a pancreatic islet of a donor; (b) expanding the stem cell exvivo to produce a progenitor cell; and (c) transferring the progenitorcell into the patient, wherein the progenitor cell differentiates into ahepatocyte.
 75. The method of claim 74, wherein said nestin-positivepancreatic stem cell is also ABCG2-positive.
 76. The method of claim 74,wherein said nestin-positive pancreatic stem cell is also positive forat least one of the markers selected from the group consisting ofOct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1, Nestin, Integrinsubunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.
 77. The method ofclaim 74, wherein said nestin positive pancreatic stem cell does notexpress at least one of the markers selected from the group consistingof CD34, CD45, CD133, MHC class I and MHC class II.
 78. A method oftreating a patient with liver disease, comprising the steps of: (a)isolating an ABCG2-positive pancreatic stem cell from a pancreatic isletof a donor; (b) expanding the stem cell ex vivo to produce a progenitorcell; and (c) transferring the progenitor cell into the patient, whereinthe progenitor cell differentiates into a hepatocyte.
 79. The method ofclaim 74 or 78, wherein the patient serves as the donor for said stemcells of step a.
 80. A method of treating a patient with liver disease,comprising the steps of: (a) isolating a nestin-positive pancreatic stemcell from a pancreatic islet of a donor; (b) differentiating the stemcell ex vivo to produce a hepatocyte; and (c) transferring thehepatocyte into the patient.
 81. The method of claim 80, wherein saidnestin-positive pancreatic stem cell is also ABCG2-positive.
 82. Themethod of claim 80, wherein said nestin-positive pancreatic stem cell isalso positive for at least one of the markers selected from the groupconsisting of Oct3/4, GLP-1 receptor, latrophilin (type 2), Hes-1,Nestin, Integrin subunits α6 and β1, C-kit, MDR-1, SUR-1, or Kir 6.2.83. The method of claim 80, wherein said nestin positive pancreatic stemcell does not express at least one of the markers selected from thegroup consisting of CD34, CD45, CD133, MHC class I and MHC class II. 84.A method of treating a patient with liver disease, comprising the stepsof: (a) isolating an ABCG2-positive pancreatic stem cell from apancreatic islet of a donor; (b) differentiating the stem cell ex vivoto produce a hepatocyte; and (c) transferring the hepatocyte into thepatient.
 85. The method of claim 80 or 84, wherein the patient serves asthe donor for said stem cells of step a.
 86. A pharmaceuticalcomposition comprising the isolated stem cell of claim 45 admixed with aphysiologically compatible carrier.
 87. A pharmaceutical compositioncomprising the isolated stem cell of claim 46 admixed with aphysiologically compatible carrier.
 88. A pharmaceutical compositioncomprising the isolated stem cell of claim 49 admixed with aphysiologically compatible carrier.